Combination therapy for cancer treatment

Abstract

The present invention relates to the field of binding molecules. In particular, the present invention relates to the field of therapeutic binding molecules for the treatment of diseases involving abnormal cells, such as cancer cells. In particular, the present invention relates to multispecific antibodies that bind to the extracellular portion of two or more different membrane-associated proteins, thereby modulating the biological activity expressed by the cells, and the use of such antibodies in combination therapy.

Description

Technical Field

[0001] The present invention relates to the field of binding molecules. Specifically, the present invention relates to the field of therapeutic binding molecules for the treatment of diseases including abnormal cells such as cancer cells. In particular, the present invention relates to multispecific antibodies that bind to the extracellular portions of two or more different membrane-bound proteins, thereby modulating the biological activities expressed by cells, and to the use of such antibodies in combination therapies.

Background Art

[0002] Cancer remains a major cause of death worldwide, despite many advances in the treatment of disease and increased knowledge of the molecular events leading to cancer.

[0003] Conventionally, the discovery of most cancer drugs has focused on agents that block essential cell functions and kill dividing cells. However, in the case of advanced cancer, no matter how aggressively applied, even to the point where patients experience life-threatening side effects from the treatment, chemotherapy rarely results in a complete cure. In most cases, tumors in patients only stop growing or shrink temporarily (termed remission), and again, in some cases, start growing more rapidly (termed recurrence), making treatment increasingly difficult. More recently, the focus of cancer drug development has shifted from widespread cytotoxic chemotherapy to less toxic targeted cell inhibitory therapies. Treatment of advanced cancer has been clinically validated in leukemia and some other cancers. However, it remains proven that in most cancer tumors, the targeted approach is not effective enough to completely eliminate cancer in most patients.

[0004] Cancer targeting has been achieved using a variety of different methods, including, for example, small molecules directed at signaling proteins on which cancer depends for survival and / or growth; vaccines containing tumor-specific proteins; cell therapies including antibodies that target tumors with immune cells and cytotoxic molecules that actively kill tumor cells; and disrupting signaling and / or (re)directing the host immune system to tumor cells.

[0005] (Re)direction of the immune system can be achieved in several ways. One way is by activating T cell costimulatory molecules such as the tumor necrosis factor receptor superfamily, including CD137 (4-1BB, TNFRSF9). Activation of CD137 leads to increased T cell proliferation, cytokine production, and CD8 + This results in extended T cell survival. Another mechanism involves blocking negative signals induced by molecules involved in immune checkpoints, such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed cell death (PD-1) expressed on T cells, or its homologous ligand, programmed cell death 1 ligand 1 (PD-L1), which may be expressed on tumor cells. Upon binding of PD-L1 to PD-1, the signaling leads to attenuation of T cell receptor (TCR) signaling and T cell depletion. This is a mechanism used by tumors to evade and / or suppress the immune system.

[0006] This immunosuppression can be blocked by immune checkpoint inhibitor therapy (ICI), such as antagonistic antibodies against PD-1 or PD-L1. ICI therapy involves activated CD8 + A subset of cancer patients correlated with T-cell infiltration and proliferation have demonstrated a remarkably sustained response. While ICI combinations (e.g., anti-PD-1 and anti-CTLA-4) have been shown to further enhance efficacy, they come at the cost of toxicity, with the majority of patients experiencing grade 3 or 4 treatment-related adverse events.

[0007] Dual targeting of the PD-1 / PD-L1 axis and CD137 may be beneficial for optimally engaging specific antitumor immunity. Currently, the two most advanced therapeutic CD137 agonist antibodies in clinical trials are urerumab (IgG4) and utomirumab (IgG2). Development of urerumab has been halted due to dose-dependent hepatitis resulting from systemic activation of the CD137 pathway in patients. Dose reduction is necessary for safe administration of urerumab, and 0.1 mg / kg was selected for combination studies with PD-1 inhibitors. Utomirumab is better tolerated by patients, but has modest antitumor activity as monotherapy and does not show a clear synergistic effect with PD-1 blockade in combination therapy.

[0008] Therefore, there remains a need to provide healthcare professionals treating cancer, particularly advanced or metastatic solid tumors, with more and better options. [Overview of the Initiative]

[0009] This disclosure relates to a combination therapy for cancer in patients requiring cancer treatment, in which a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein is used together with a PD-L1 or PD-1 inhibitor.

[0010] This disclosure provides means and methods for (re)directing immune system components in the treatment of cancer, particularly advanced or metastatic solid tumors.

[0011] In certain embodiments, the Disclosure provides a multispecific antibody for use in a method of treating cancer in a subject requiring cancer treatment, comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, wherein the treatment further comprises administering a PD-L1 or PD-1 inhibitor.

[0012] In certain embodiments, the Disclosure provides a multispecific antibody for use in a method of treating cancer in subjects requiring cancer treatment, comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, wherein the multispecific antibody is intended for simultaneous, sequential, or separate administration with a PD-1 or PD-L1 inhibitor.

[0013] In certain embodiments, the Disclosure provides a combination of a multispecific antibody, comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, and a PD-L1 or PD-1 inhibitor, for use in a method of treating cancer in subjects requiring cancer treatment.

[0014] In certain embodiments, the Disclosure provides a method for treating cancer in a subject requiring cancer treatment, comprising administering to the subject requiring cancer treatment a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, and a PD-1 or PD-L1 inhibitor.

[0015] In certain embodiments, multispecific antibodies are administered simultaneously with, sequentially with, or separately from, PD-1 or PD-L1 inhibitors.

[0016] In certain embodiments, cancer is an advanced or metastatic solid tumor, such as NSCLC, selected from locally advanced or metastatic lung cancer and locally advanced or metastatic melanoma. In certain embodiments, melanoma is selected from cutaneous melanoma, acral melanoma, or mucosal melanoma. In certain embodiments, cancer is Merkel cell carcinoma (also known as neuroendocrine carcinoma or cord-like carcinoma of the skin).

[0017] In certain embodiments, the cancer recurs in response to PD-1 / PD-L1 therapy and / or is positive for PD-L1 expression.

[0018] In certain embodiments, the multispecific antibody is administered before the PD-1 or PD-L1 inhibitor.

[0019] In certain embodiments, a multispecific antibody is a bispecific antibody.

[0020] In a particular embodiment, the antigen-binding site of a multispecific antibody that binds to the extracellular portion of a second membrane protein binds to PD-L1.

[0021] In certain aspects, the disclosure relates to a multispecific antibody for use in a method of treating cancer in a subject requiring cancer treatment, wherein the antibody is A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 50, CDR2 having the amino acid sequence shown in SEQ ID NO: 51, and CDR3 having the amino acid sequence shown in SEQ ID NO: 52, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 40, CDR2 having the amino acid sequence shown in SEQ ID NO: 41, and CDR3 having the amino acid sequence shown in SEQ ID NO: 42, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 21, CDR2 having the amino acid sequence shown in SEQ ID NO: 22, and CDR3 having the amino acid sequence shown in SEQ ID NO: 23, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 32, CDR2 having the amino acid sequence shown in SEQ ID NO: 33, and CDR3 having the amino acid sequence shown in SEQ ID NO: 34, comprising a binding domain that binds to CD137, and / or Antibodies, A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 68, CDR2 having the amino acid sequence shown in SEQ ID NO: 55, and CDR3 having the amino acid sequence shown in SEQ ID NO: 56, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 93, CDR2 having the amino acid sequence shown in SEQ ID NO: 94, and CDR3 having the amino acid sequence shown in SEQ ID NO: 95, or A variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 93, a CDR2 having the amino acid sequence shown by SEQ ID NO: 101, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 102, or a variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 90, a CDR2 having the amino acid sequence shown by SEQ ID NO: 79, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 91, and comprising a binding domain that binds to PD-L1, each of the individual SEQ ID NOs has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, or substitutions, or combinations thereof, Provided is a multispecific antibody wherein the multispecific antibody is for simultaneous, sequential, or separate administration with a PD-1 or PD-L1 inhibitor.

[0022] In certain embodiments, the disclosure provides a method of treating cancer in a subject that needs treatment for cancer, the method comprising administering a multispecific antibody that comprises a variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 50, a CDR2 having the amino acid sequence shown by SEQ ID NO: 51, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 52, or a variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 40, a CDR2 having the amino acid sequence shown by SEQ ID NO: 41, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 42, or a variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 21, a CDR2 having the amino acid sequence shown by SEQ ID NO: 22, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 23, or a variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 32, a CDR2 having the amino acid sequence shown by SEQ ID NO: 33, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 34, and comprising a binding domain that binds to CD137, and / or the antibody A variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 68, a CDR2 having the amino acid sequence shown by SEQ ID NO: 55, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 56, or A variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 93, a CDR2 having the amino acid sequence shown by SEQ ID NO: 94, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 95, or A variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 93, a CDR2 having the amino acid sequence shown by SEQ ID NO: 101, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 102, or A variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 90, a CDR2 having the amino acid sequence shown by SEQ ID NO: 79, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 91, comprising a binding domain that binds to PD-L1, A multispecific antibody in which each of the individual SEQ ID NOs has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, or substitutions, or combinations thereof, and A PD-1 or PD-L1 inhibitor, administering to a subject in need of treatment for cancer, a method comprising.

[0023] In certain embodiments, the present disclosure is a multispecific antibody for use in a method of treating cancer in a subject in need of treatment for cancer, wherein the antibody is A variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 50, a CDR2 having the amino acid sequence shown by SEQ ID NO: 51, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 52, or A variable domain comprising a CDR1 having the amino acid sequence shown by SEQ ID NO: 40, a CDR2 having the amino acid sequence shown by SEQ ID NO: 41, and a CDR3 having the amino acid sequence shown by SEQ ID NO: 42, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 21, CDR2 having the amino acid sequence shown in SEQ ID NO: 22, and CDR3 having the amino acid sequence shown in SEQ ID NO: 23, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 32, CDR2 having the amino acid sequence shown in SEQ ID NO: 33, and CDR3 having the amino acid sequence shown in SEQ ID NO: 34, comprising a binding domain that binds to CD137, and / or Antibodies, A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 68, CDR2 having the amino acid sequence shown in SEQ ID NO: 55, and CDR3 having the amino acid sequence shown in SEQ ID NO: 56, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 93, CDR2 having the amino acid sequence shown in SEQ ID NO: 94, and CDR3 having the amino acid sequence shown in SEQ ID NO: 95, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 93, CDR2 having the amino acid sequence shown in SEQ ID NO: 101, and CDR3 having the amino acid sequence shown in SEQ ID NO: 102, or It includes a variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 90, CDR2 having the amino acid sequence shown in SEQ ID NO: 79, and CDR3 having the amino acid sequence shown in SEQ ID NO: 91, and a binding domain that binds to PD-L1. Each of the individual sequence numbers has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, or substitutions, or combinations thereof. The multispecific antibody is intended for simultaneous, subsequent, or separate administration with a PD-1 or PD-L1 inhibitor. This invention provides multispecific antibodies for cancer that is progressive or metastatic solid tumors.

[0024] In certain embodiments, the Disclosure provides a drug combination or kit of parts, comprising a multispecific antibody having an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, and instructions for the use of the multispecific antibody in combination with a PD-1 or PD-L1 inhibitor, particularly for the treatment of cancer.

[0025] In certain embodiments, the Disclosure provides a combination of a multispecific antibody, comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of PD-L1, and a PD-1 or PD-L1 inhibitor, for use in a method of treating cancer in subjects requiring cancer treatment.

[0026] In certain embodiments, the Disclosure provides a PD-1 or PD-L1 inhibitor for the treatment of cancer in subjects requiring cancer treatment, wherein the PD-L1 inhibitor is for simultaneous or sequential administration with a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein.

[0027] In certain embodiments, the Disclosure provides a multispecific antibody for use in the treatment of cancer in subjects who have been or are scheduled to be administered a PD-1 or PD-L1 inhibitor, comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of PD-L1.

[0028] In certain embodiments, the Disclosure provides a PD-1 or PD-L1 inhibitor for use in the treatment of cancer in patients who have been or are scheduled to be administered a multispecific antibody, comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of PD-L1.

[0029] In certain embodiments, the multispecific antibody is administered in doses of 25-300 mg, such as 25-150 mg or 25-100 mg, or 25-50 mg or 50-100 mg.

[0030] In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in doses of 200-600 mg, such as 300-500 mg or approximately 400 mg.

[0031] In certain aspects, cancer is a progressive or metastatic solid tumor.

[0032] In certain embodiments, the multispecific antibody, PD-L1, or PD-1 inhibitor is an isolated antibody.

[0033] In certain aspects, the subject is a human subject.

[0034] In certain embodiments, the subjects have not yet received prior treatment with a CD137 agonist, prior treatment with CAR T-cell therapy, or prior treatment including targeted small molecule therapy or radiotherapy. [Modes for carrying out the invention]

[0035] In certain embodiments, the Disclosure provides a multispecific antibody for use in a method of treating cancer in a subject requiring treatment for cancer, comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, wherein the multispecific antibody is for simultaneous, sequential, or separate administration with a PD-1 or PD-L1 inhibitor. In certain embodiments, the Disclosure provides a method of treating cancer in a subject requiring treatment for cancer, comprising administering a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, and a PD-1 or PD-L1 inhibitor to the subject.

[0036] In a particular embodiment, the second membrane protein is PD-L1, and the antigen-binding site that binds to the extracellular portion of the second membrane protein binds to PD-L1.

[0037] CD137 can be expressed by activated T cells. CD137 is also found in other cells such as dendritic cells, natural killer cells, granulocytes, and cells of the blood vessel wall at inflammatory sites. This protein is known for its costimulatory activity for T cell activation. CD137 is known by many different names, including: TNFRSF9; TNF receptor superfamily member 9; tumor necrosis factor receptor superfamily member 9; T cell antigen 4-1BB homolog; 4-1BB ligand receptor; T cell antigen ILA; CD137 antigen; CDw137; ILA; interleukin-activated receptor, mouse Ly63 homolog; lymphocyte activation-induced (ILA); mouse 4-1BB homolog; receptor protein 4-1BB; cell antigen ILA; and 4-1BB. The external IDs for CD137 are HGNC:11924, Entrez Gene:3604, Ensembl:ENSG00000049249, OMIM:602250, and UniProtKB:Q07011. CD137 is the most commonly upregulated inductive receptor on activated CD8+ T cells. CD137 signaling enhances T cell function by activating NF-κB [Arch et al, 1998]. CD4+ T cells, monocytes, B cells, dendritic cell (DC) subpopulations, and other cellular immune cell types, including granulocytes and NK cells, can express CD137 at varying levels [Shao et al, 2011]. In monocytes, CD137 is inducible by activation with lipopolysaccharide (LPS) and IL-1b. In B lymphocytes, CD137 expression is induced by transformation with antibodies against cell surface immunoglobulins and EBV. In DCs, CD137 ligation induces maturation through the upregulation of B7 costimulatory molecules (CD80 and CD86), in addition to improving the production and viability of inflammatory cytokines (IL-6 and IL-12) [Makkouk et al, 2015]. The innate function of CD137 ligation to neutrophils is increased phagocytosis in bacterial and parasitic infections.In addition, CD137 ligation blocks IL-3 / IL-5 / GM-CSF receptor-mediated anti-apoptotic signaling in neutrophils and eosinophils in vitro, thereby preventing granulocyte accumulation [Simon, 2001; Vinay et al, 2011]. In non-lymphoid cells such as chondrocytes, endothelial cells, and tumor cells, CD137 expression is driven by cytokine stimulation, including IL-1β in chondrocytes, the inflammatory cytokine TNF-alpha / IFNγ / endothelial IL-1β in endothelial cells, and IFNγ in tumor cells. The ligand that stimulates CD137 (CD137L) is expressed on activated antigen-presenting cells. CD137 exists intramembrane as monomers and dimers [Pollok et al, 1993].

[0038] The B7 family comprises numerous structurally related cell surface proteins that bind to receptors on lymphocytes that modulate the immune response. Lymphocyte activation is initiated by the involvement of cell surface, antigen-specific T cell receptors, or B cell receptors. Additional signals delivered simultaneously by B7 ligands further determine the immune response of these cells. These so-called "co-stimulatory" or "co-inhibitory" signals are delivered by B7 family members via the CD28 family of receptors on lymphocytes. Binding of B7 family members to costimulatory receptors enhances the immune response, while binding to co-inhibitory receptors attenuates the immune response. Currently, the following members are considered to be part of this family: B7.1 (CD80), B7.2 (CD86), inducible costimulatory ligand (ICOS-L), programmed death-1 ligand (PD-L1), programmed death-2 ligand (PD-L2), B7-H3 (CD276), B7-H4, B7-H5, B7-H6, and B7-H7. B7 family members are expressed in lymphoid and non-lymphoid tissues. The effects of these members on regulating the immune response have been demonstrated in the development of immunodeficiency and autoimmune diseases in mice with mutations in B7 family genes. Manipulation of signals delivered by B7 ligands has shown potential in the treatment of autoimmune, inflammatory, and cancer diseases.

[0039] PD-L1 is a type 1 transmembrane protein that plays a role in suppressing the immune response during certain events, such as pregnancy, tissue allografts, autoimmune diseases, and other disease conditions such as hepatitis. PD-L1 is expressed in various types of cancer, particularly NSCLC (Boland et al., 2013, Velcheti et al., 2014), melanoma, renal cell carcinoma, gastric cancer, hepatocyte cancer, and various leukemias and multiple myeloma (Bernstein et al., 2014, Thompson et al., 2005). PD-L1 is present in the cytoplasm and plasma membrane of cancer cells, but not all cancers or all cells within a tumor express PD-L1 (Dong et al., 2002). Several tumor microenvironment cells contribute to immunosuppression by upregulating PD-L1 expression. This effect is called "adaptive immune resistance" because tumors protect themselves by inducing PD-L1 in response to IFN-γ produced by activated T cells (Sharma et al., 2017). PD-L1 can also be regulated by oncogenes, and this mechanism is known as intrinsic immune resistance (Akbay et al., 2013). Within the tumor microenvironment, PD-L1 is also expressed on bone marrow cells and activated T cells (Tumeh et al., 2014). PD-L1 expression is induced by several pro-inflammatory molecules, including type I and type II IFN-γ, TNF-α, LPS, GM-CSF, and VEGF, as well as cytokines IL-10 and IL-4, with IFN-γ being the most potent inducer (Sznol and Chen, 2013).

[0040] Programmed cell death 1 protein (PD-1) is a cell surface receptor belonging to the CD28 family of receptors, expressed on T cells and pro-B cells. PD-1 is currently known to bind to two ligands, PD-L1 and PD-L2. Functioning as an immune checkpoint, PD-1 plays a crucial role in downregulating the immune system by inhibiting T cell activation, thereby reducing autoimmunity and promoting self-tolerance. The inhibitory effect of PD-1 is thought to be achieved through a dual mechanism: promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells). PD-1 is known by numerous different names, including PDCD1, programmed cell death 1, systemic lupus erythematosus sensitivity 2, protein PD-1, HPD-1, PD1, programmed cell death 1 protein, CD279 antigen, CD279, HPD-L, HSLE1, SLEB2, and PD-1. External IDs for PD-1 are HGNC:8760, Entrez Gene:5133, Ensembl:ENSG00000188389, OMIM:600244, and UniProtKB:Q15116. A new class of drugs, PD-1 inhibitors, which block the activity of PD-1, activate the immune system to attack tumors and have therefore been successfully used to treat several types of cancer.

[0041] The binding of PD-L1 to PD-1 or B7.1(CD80) transmits an inhibitory signal that reduces the proliferation of PD-1-expressing T cells. PD-1 is thought to be able to regulate the accumulation of exogenous antigen-specific T cells through apoptosis. PD-L1 is expressed by various cancer cells, and its expression is thought to be at least partially involved in the attenuation of the immune response against cancer cells. PD-L1 is a member of the B7 family of proteins and is known by various other names such as CD274 molecule, CD274 antigen, B7 homolog 1, PDCD1 ligand 1, PDCD1LG1, PDCD1L1, B7H1, PDL1, programmed cell death 1 ligand 1, programmed death ligand 1, B7-H1, and B7-H. The external IDs for CD274 are HGNC:17635;Entrez gene:29126, Ensembl:ENSG00000120217, OMIM:605402, and UniProtKB:Q9NZQ7.

[0042] PD-L2 is the secondary ligand for PD-1. PD-L2-mediated involvement of PD-1 inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2 / PD-1 binding inhibits B7-CD28 signaling. At high antigen concentrations, PD-L2 / PD-1 binding reduces cytokine production. PD-L expression is upregulated on antigen-presenting cells by interferon-gamma treatment. It is expressed in several normal tissues and various tumors. PD-L1 and PD-L2 are thought to have overlapping functions and regulate T cell responses. The protein is known by several other names, including programmed cell death 1 ligand 2, B7 dendritic cell molecule, programmed cell death ligand 2, butyrophyllin B7-DC, PDCD1 ligand 2, PD-1 ligand 2, PDCD1L2, B7-DC, CD273, B7DC, PDL2, PD-1-ligand 2, CD273 antigen, BA574F11.2, and Btdc. The external IDs for PD-L2 are HGNC:18731, Entrez Gene:80380, Ensembl:ENSG00000197646, OMIM:605723, and UniProtKB:Q9BQ51.

[0043] PD-1 inhibitors are well known in the art. These include antibodies that bind to and block PD-1. Pembrolizumab is a humanized antibody used in cancer immunotherapy to treat melanoma, lung cancer, head and neck cancer, Hodgkin lymphoma, gastric cancer, and cervical cancer. It is administered by slow intravenous injection. Pembrolizumab is a therapeutic antibody that binds to and blocks PD-1 located on lymphocytes. This receptor is a so-called "immune checkpoint" and is therefore generally responsible for preventing the immune system from attacking the body's own tissues. Normally, the PD-1 receptor on activated T cells binds to PD-L1 or PD-L2 ligands present on normal cells in the body, inactivating any potential cell-mediated immune response against these cells. Many cancers produce proteins such as PD-L1 that also bind to the PD-1 receptor and therefore block the body's ability to kill cancer. Pembrolizumab works by inhibiting the lymphocyte PD-1 receptor, blocking its ligand which would inactivate it and prevent an immune response. This allows the immune system to target and destroy cancer cells, but also blocks a crucial mechanism that prevents the immune system from attacking the body itself.

[0044] Tumors often have mutations that cause DNA mismatch repair failure. This, in turn, often leads to microsatellite instability, causing the tumor to produce numerous mutant proteins that can function as tumor antigens, triggering an immune response against the tumor. By preventing the self-checkpoint system from blocking T cells,

[14]

[30] pembrolizumab appears to facilitate the immune system's clearance of any such tumor.

[0045] Pembrolizumab was approved for medical use in the United States in 2014. In 2017, the U.S. Food and Drug Administration (FDA) approved pembrolizumab for any unresectable or metastatic solid tumors with certain genetic abnormalities (mismatch repair failure or microsatellite instability).

[0046] In certain embodiments, the multispecific antibody or method used in this disclosure binds to a second membrane protein that is not a member of the TNF receptor superfamily. In certain embodiments, the second membrane protein is a member of the B7 family. In certain embodiments, the second membrane protein is PD-L1 or PD-L2, and in certain embodiments, it is PD-L1.

[0047] In certain embodiments, the multispecific antibody or method used in this disclosure includes a single antigen-binding site that binds to the PD-1 binding domain of PD-L1.

[0048] In certain embodiments, the multispecific antibody or method used in this disclosure binds to a second membrane protein that is not significantly expressed by T cells.

[0049] In certain embodiments, the multispecific antibody or method used in this disclosure binds to a second membrane protein expressed on antigen-presenting cells, tumor cells, virus-infected cells, or parasite-infected cells.

[0050] In certain embodiments, a multispecific antibody or method using the present disclosure binds to a second membrane protein, which is a membrane protein located in one or more zones on the cell membrane. In certain embodiments, the zones are clusters, domains, microdomains, or compartments on the cell membrane, and in certain embodiments, immunological synapses.

[0051] In certain embodiments, the multispecific antibody or method used in this disclosure binds to a second membrane protein that is present on the cell membrane as part of a multimeric membrane protein comprising two or more of the second membrane proteins. In certain embodiments, the second membrane protein is present on the cell membrane as part of a homodimer or homotrimer.

[0052] In certain embodiments, the multispecific antibody or method used by the Disclosure comprises a single antigen-binding site that binds to the CD137L-binding domain of CD137.

[0053] In certain embodiments, a multispecific antibody or method using the present disclosure includes a single antigen-binding site that blocks the binding of a ligand to CD137 or, in certain embodiments, a site that binds to an extracellular ligand-blocking site of CD137, or to a CD137L-blocking site.

[0054] In certain embodiments, a multispecific antibody or method using the Disclosure includes, in the case of a bivalent monospecific antibody format containing two of the variable domains that bind to CD137, a variable domain that either does not stimulate the activity of CD137 on a cell, or stimulates it at a reduced level compared to one of the variable domains as part of a multispecific antibody having a second variable domain that binds to a tumor-associated antigen such as a member of the B7 family or PD-L1.

[0055] In certain embodiments, a variable domain that binds to the extracellular portion of CD137 is defined as a variable domain that, when present in a bivalent monospecific antibody format (such as IgG, particularly IgG1, or human IgG1) containing two of the variable domains that bind to CD137, either does not stimulate the activity of CD137 on the cell, or stimulates it at a reduced level compared to one of the variable domains as part of a bispecific antibody (such as IgG, particularly IgG1, or human IgG1) having a second variable domain that binds to a tumor-associated antigen. Those skilled in the art will see that when comparing the CD137 stimulating activity between the bivalent monospecific and bispecific antibodies mentioned above, the antibodies are in the same format (IgG, particularly IgG1 or human IgG1, etc.), and the difference between the antibodies being compared is, on the one hand, a monospecific antibody with the presence of identical variable domain binding in a bivalent form that binds to CD137, and on the other hand, one of the CD137 variable domains and MF5554 (SEQ ID NO: 53), MF5576 (SEQ ID NO: 57), MF5578 (SEQ ID NO: 59), MF9375 (SEQ ID NO: 62), MF9376 (SEQ ID NO: 64), MF7702 (SEQ ID NO: 67), It will be recognized that these are bispecific antibodies containing a variable domain that binds to tumor-associated antigens, such as the variable domain containing the VH amino acid sequence of MF5359 (SEQ ID NO: 69), MF5377 (SEQ ID NO: 73), MF5382 (SEQ ID NO: 77), MF5424 (SEQ ID NO: 81), MF5426 (SEQ ID NO: 85), MF5439 (SEQ ID NO: 89), MF5442 (SEQ ID NO: 92), MF5553 (SEQ ID NO: 96), MF5557 (SEQ ID NO: 97), MF5561 (SEQ ID NO: 100), MF5576 (SEQ ID NO: 103), MF5594 (SEQ ID NO: 104), or MF5708 (SEQ ID NO: 107). In certain embodiments, when in a bivalent monospecific antibody format, the variable domain that binds to the non-irritating extracellular portion of CD137 includes a VH CDR sequence such as MF6754, MF6808, MF6763, MF6785, or MF6797.

[0056] In certain embodiments, a multispecific antibody or method using the present disclosure includes a variable domain that binds to the extracellular portion of CD137, which, when combined with a second variable domain that binds to PD-L1 in the presence of a first cell expressing CD137 and a second cell expressing PD-L1, can stimulate the activity of CD137 on the cell.

[0057] In certain embodiments, the multispecific antibodies or methods used in this disclosure can simultaneously conjugate CD137 and PD-L1.

[0058] The multispecific antibodies according to this disclosure, which bind to CD137 and a second membrane protein, particularly the extracellular portion of a membrane protein that is a member of the B7 family, offer the advantage of being able to particularly promote a desired immune response because B7 family members deliver "co-stimulatory" or "co-inhibitory" signals to lymphocytes, thereby enhancing or attenuating the immune response. Therefore, by targeting a second transmembrane protein, particularly a transmembrane protein that is a member of the B7 family, it is possible to enhance stimulatory signals and / or inhibit inhibitory signals, thereby inducing or enhancing a desired immune response against abnormal cells, such as cancer cells. As a result, the multispecific antibodies according to this disclosure can be used in cancer treatment in subjects requiring cancer treatment, where a desired immune response is induced against abnormal cells found in cancer.

[0059] In certain embodiments, the multispecific antibody according to the Disclosure has one antigen-binding site that can bind to the extracellular portion of CD137 and a second antigen-binding site that can bind to the extracellular portion of a second membrane protein, which in certain embodiments is not a member of the TNF receptor superfamily but is a member of the B7 family or, in particular embodiments, PD-L1, etc. This offers the advantage of at least partially avoiding cis-activation of (immune) cells, such as T cells expressing several different members of the TNF receptor superfamily, thereby reducing potential adverse side effects and toxicity resulting from nonspecific T cell activation. The prior art approach may result in cis-T cell activation, meaning the absence of a second target, and may carry the risk of an excessive T cell response, for example, leading to a cytokine storm. Consequently, such prior art approaches have an increased potential for adverse side effects compared to the binding molecule according to the Disclosure, which has an antigen-binding site that can bind to CD137 and an antigen-binding site that can bind to the extracellular portion of a second membrane protein.

[0060] In certain embodiments, the disclosure relates to a multispecific antibody that targets both PD-L1 and CD137 in order to partially evade cis-activation of (immune) cells such as T cells. In certain embodiments, the variable domains that bind to the extracellular portion of CD137 are domains that, when in a bivalent monospecific antibody format containing two of such CD137-binding domains, do not stimulate the activity of CD137 on cells, or stimulate it at a reduced level compared to one of such variable domains as part of a multispecific antibody having a second variable domain that binds to a tumor-associated antigen, in certain embodiments a member of the B7 family, or PD-L1, etc. Preferred CD137-binding arms are disclosed in WO2018 / 056821.

[0061] In certain embodiments, the multispecific antibody or method used in this disclosure may be an agonistic CD137 antibody, for example, an antibody capable of stimulating the activity of CD137. In certain embodiments, the multispecific antibody or method used in this disclosure may be an antagonistic CD137 antibody, for example, an antibody capable of reducing the activity of CD137.

[0062] In certain embodiments, the multispecific antibody or method used in this disclosure may be an agonistic B7 antibody, for example, an antibody capable of stimulating the activity of B7. In certain embodiments, the multispecific antibody or method used in this disclosure may be an antagonistic B7 antibody, for example, an antibody capable of reducing the activity of a B7 family member.

[0063] In certain embodiments, the multispecific antibody or method used in this disclosure may be an agonistic PD-L1 antibody, for example, an antibody capable of stimulating the activity of PD-L1. In certain embodiments, the multispecific antibody or method used in this disclosure may be an antagonistic PD-L1 antibody, for example, an antibody capable of reducing the activity of PD-L1.

[0064] In certain embodiments, a multispecific antibody or method used in this disclosure may stimulate CD137 activity when bound to a B7 family member. In certain embodiments, a multispecific antibody stimulates CD137 activity when bound to both CD137 and PD-L1. In certain embodiments, a multispecific antibody induces or activates CD137 signaling only in the presence of PD-L1 expressing cells.

[0065] In certain embodiments, a multispecific antibody or method using the present disclosure includes an antigen-binding site comprising one immunoglobulin variable domain that binds to CD137 and one immunoglobulin variable domain that binds to the extracellular portion of a second membrane protein.

[0066] In certain embodiments, a variable domain included by a multispecific antibody or method using the Disclosure that binds to the extracellular portion of CD137 and at least partially blocks the binding of a CD137 ligand to CD137 is a variable domain comprising the VH amino acid sequence of MF6783 (SEQ ID NO: 1), MF6861 (SEQ ID NO: 5), MF6795 (SEQ ID NO: 9), MF6808 (SEQ ID NO: 13), MF6798 (SEQ ID NO: 17), MF6754 (SEQ ID NO: 20), MF6763 (SEQ ID NO: 24), MF6744 (SEQ ID NO: 28), MF6785 (SEQ ID NO: 31), MF6825 (SEQ ID NO: 35), MF6737 (SEQ ID NO: 39), MF6749 (SEQ ID NO: 43), MF6788 (SEQ ID NO: 46), or MF6797 (SEQ ID NO: 49).

[0067] In certain embodiments, a variable domain included by a multispecific antibody or method using the Disclosure that binds to the extracellular portion of CD137 and at least partially blocks the binding of a CD137 ligand to CD137 is a variable domain comprising the CDR1, CDR2, and CDR3 amino acid sequences of the VH of MF6783 (SEQ ID NO: 1), MF6861 (SEQ ID NO: 5), MF6795 (SEQ ID NO: 9), MF6808 (SEQ ID NO: 13), MF6798 (SEQ ID NO: 17), MF6754 (SEQ ID NO: 20), MF6763 (SEQ ID NO: 24), MF6744 (SEQ ID NO: 28), MF6785 (SEQ ID NO: 31), MF6825 (SEQ ID NO: 35), MF6737 (SEQ ID NO: 39), MF6749 (SEQ ID NO: 43), MF6788 (SEQ ID NO: 46), or MF6797 (SEQ ID NO: 49).

[0068] In certain embodiments, a multispecific antibody, or a functional portion, derivative, and / or analog thereof, or method by use of the present disclosure comprising a variable domain that binds to the extracellular portion of CD137, includes a heavy chain variable region having a CDR3 region comprising the amino acid sequence of the CDR3 region of the variable heavy chain region of MF6808 (SEQ ID NO: 13), MF6754 (SEQ ID NO: 23), MF6763 (SEQ ID NO: 27), MF6785 (SEQ ID NO: 34), or MF6797 (SEQ ID NO: 52).

[0069] In certain embodiments, a multispecific antibody, or a functional portion, derivative, and / or analog thereof, or method by use of the present disclosure comprising a variable domain that binds to the extracellular portion of CD137, includes a heavy chain variable region having a CDR2 region comprising the amino acid sequence of the CDR2 region of the variable heavy chain region of MF6808 (SEQ ID NO: 13), MF6754 (SEQ ID NO: 22), MF6763 (SEQ ID NO: 26), MF6785 (SEQ ID NO: 33), or MF6797 (SEQ ID NO: 51).

[0070] In certain embodiments, a multispecific antibody, or a functional portion, derivative, and / or analog thereof, or method by use of the present disclosure comprising a variable domain that binds to the extracellular portion of CD137, includes a heavy chain variable region having a CDR1 region comprising the amino acid sequence of the CDR1 region of the variable heavy chain region of MF6808 (SEQ ID NO: 13), MF6754 (SEQ ID NO: 21), MF6763 (SEQ ID NO: 25), MF6785 (SEQ ID NO: 32), or MF6797 (SEQ ID NO: 50).

[0071] In certain embodiments, a multispecific antibody, or a functional portion, derivative, and / or analog thereof, or method by use of the present disclosure comprising a variable domain that binds to the extracellular portion of CD137, includes a heavy chain variable region having CDR1, CDR2, and CDR3 regions, which include the amino acid sequences of CDR1, CDR2, and CDR3 of one of the variable heavy chain regions of VH presented as MF6808, MF6754, MF6763, MF6785, or MF6797. In certain embodiments, the CDR1, CDR2, and CDR3 sequences are selected from the same VH region.

[0072] In certain embodiments, a multispecific antibody, or functional portion thereof, derivative, and / or analogue, or method by use of the Disclosure comprising a variable domain that binds to the extracellular portion of CD137, includes the amino acid sequence of the variable heavy chain region of MF6808, MF6754, MF6763, MF6785, or MF6797, having up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of the VH of the indicated MF. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, 0, 1, or 2, or 0 or 1, relative to the amino acid sequence of the VH of the indicated MF. In certain embodiments, amino acid insertions, deletions, substitutions, or combinations thereof, if present, are not present in the amino acid sequence of the CDR region.

[0073] In certain embodiments, a variable domain, functional portion thereof, derivatives, and / or analogues included by the use of the multispecific antibody or method of the present disclosure, which binds to the extracellular portion of CD137, include a VH region having the amino acid sequence of CDR3, or a VH region having the amino acid sequences of CDR1, CDR2, and CDR3 of one of the VH regions of MF6808, MF6754, MF6763, MF6785, or MF6797. In certain embodiments, the variable domain that binds to the extracellular portion of CD137 includes a VH region having the VH amino acid sequence of MF6808 (SEQ ID NO: 13), MF6754 (SEQ ID NO: 20), MF6763 (SEQ ID NO: 24), MF6785 (SEQ ID NO: 31), or MF6797 (SEQ ID NO: 49), with up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, or 10, or 0, 1, 2, 3, 4, or 5, relative to the VH amino acid sequence of the indicated MF. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, or 0, 1, or 2, or 0 or 1, relative to the VH amino acid sequence of the indicated MF. In certain embodiments, amino acid insertions, deletions, substitutions, or combinations thereof, if present, are not present in the amino acid sequence of the CDR region.

[0074] In certain embodiments, a multispecific antibody, or a functional portion thereof, derivative, and / or analogue thereof, or a variable domain included by the method, which binds to the extracellular portion of PD-L1 and blocks the binding of PD-1 to PD-L1, is MF5554 (SEQ ID NO: 53), MF5576 (SEQ ID NO: 57), MF5578 (SEQ ID NO: 59), MF9375 (SEQ ID NO: 62), MF9376 (SEQ ID NO: 64), MF7702 (SEQ ID NO: 67), MF5359 (SEQ ID NO: 53). 69) A variable domain comprising the amino acid sequence of VH of MF5377 (SEQ ID NO: 73), MF5382 (SEQ ID NO: 77), MF5424 (SEQ ID NO: 81), MF5426 (SEQ ID NO: 85), MF5439 (SEQ ID NO: 89), MF5442 (SEQ ID NO: 92), MF5553 (SEQ ID NO: 96), MF5557 (SEQ ID NO: 97), MF5561 (SEQ ID NO: 100), MF5576 (SEQ ID NO: 103), MF5594 (SEQ ID NO: 104), or MF5708 (SEQ ID NO: 107). In certain embodiments, as referred to elsewhere in this specification, the CDR from the MF sequence is determined using the Kabat numbering system.

[0075] In certain embodiments, a multispecific antibody, or a functional portion thereof, derivative, and / or analogue thereof, or a variable domain included by the method, which binds to the extracellular portion of PD-L1 and blocks the binding of PD-1 to PD-L1, is MF5554 (SEQ ID NO: 53), MF5576 (SEQ ID NO: 57), MF5578 (SEQ ID NO: 59), MF9375 (SEQ ID NO: 62), MF9376 (SEQ ID NO: 64), MF7702 (SEQ ID NO: 67), MF5359 (SEQ ID NO: 69), MF537 This is a variable domain containing the CDR1, CDR2, and CDR3 sequences of the VH amino acid sequence of 7 (SEQ ID NO: 73), MF5382 (SEQ ID NO: 77), MF5424 (SEQ ID NO: 81), MF5426 (SEQ ID NO: 85), MF5439 (SEQ ID NO: 89), MF5442 (SEQ ID NO: 92), MF5553 (SEQ ID NO: 96), MF5557 (SEQ ID NO: 97), MF5561 (SEQ ID NO: 100), MF5576 (SEQ ID NO: 103), MF5594 (SEQ ID NO: 104), or MF5708 (SEQ ID NO: 107).

[0076] In certain embodiments, a multispecific antibody, or a functional portion, derivative, and / or analog thereof, or method by use of the present disclosure comprising a variable domain that binds to the extracellular portion of PD-L1, includes a heavy chain variable region having a CDR3 region comprising the amino acid sequence of the CDR3 region of the variable heavy chain region of MF5554 (SEQ ID NO: 56), MF5576 (SEQ ID NO: 58), MF5578 (SEQ ID NO: 61), MF9375 (SEQ ID NO: 56), MF9376 (SEQ ID NO: 56), MF7702 (SEQ ID NO: 56), MF5424 (SEQ ID NO: 84), MF5561 (SEQ ID NO: 102), MF5439 (SEQ ID NO: 91), MF5553 (SEQ ID NO: 56), MF5594 (SEQ ID NO: 106), MF5426 (SEQ ID NO: 88), or MF5442 (SEQ ID NO: 95).

[0077] In certain embodiments, a multispecific antibody, or a functional portion thereof, derivative, and / or analogue, or method by use of the present disclosure comprising a variable domain that binds to the extracellular portion of PD-L1, includes a heavy chain variable region having a CDR3 region comprising the amino acid sequence of the CDR3 region of the variable heavy chain region of MF7702 (SEQ ID NO: 56), MF5424 (SEQ ID NO: 84), MF5561 (SEQ ID NO: 102), MF5439 (SEQ ID NO: 91), MF5553 (SEQ ID NO: 56), MF5594 (SEQ ID NO: 106), MF5426 (SEQ ID NO: 88), or MF5442 (SEQ ID NO: 95).

[0078] In certain embodiments, a multispecific antibody, or a functional portion, derivative, and / or analog thereof, or a method using the present disclosure, includes a heavy chain variable region that binds to the extracellular portion of PD-L1, comprising a CDR3 region having the amino acid sequence shown in SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 102, or SEQ ID NO: 106. In certain embodiments, a multispecific antibody, or a functional portion, derivative, and / or analog thereof, or a method using the present disclosure, includes a heavy chain variable region that binds to the extracellular portion of PD-L1, comprising a CDR3 region having the amino acid sequence shown in SEQ ID NO: 56, SEQ ID NO: 91, SEQ ID NO: 95, or SEQ ID NO: 102, or variants thereof.

[0079] In certain embodiments, a multispecific antibody, or a functional portion, derivative, and / or analog thereof, or method by use of the present disclosure comprising a variable domain that binds to the extracellular portion of PD-L1, includes a heavy chain variable region having a CDR2 region comprising the amino acid sequence of the CDR2 region of the variable heavy chain region of MF5554 (SEQ ID NO: 55), MF5576 (SEQ ID NO: 55), MF5578 (SEQ ID NO: 3), MF9375 (SEQ ID NO: 63), MF9376 (SEQ ID NO: 66), MF7702 (SEQ ID NO: 55), MF5424 (SEQ ID NO: 83), MF5561 (SEQ ID NO: 101), MF5439 (SEQ ID NO: 79), MF5553 (SEQ ID NO: 55), MF5594 (SEQ ID NO: 105), MF5426 (SEQ ID NO: 87), or MF5442 (SEQ ID NO: 94).

[0080] In certain embodiments, a multispecific antibody, or a functional portion thereof, derivative, and / or analogue, or method by use of the present disclosure comprising a variable domain that binds to the extracellular portion of PD-L1, includes a heavy chain variable region having a CDR2 region comprising the amino acid sequence of the CDR2 region of the variable heavy chain region of MF7702 (SEQ ID NO: 56), MF5424 (SEQ ID NO: 84), MF5561 (SEQ ID NO: 102), MF5439 (SEQ ID NO: 91), MF5553 (SEQ ID NO: 56), MF5594 (SEQ ID NO: 106), MF5426 (SEQ ID NO: 88), or MF5442 (SEQ ID NO: 95).

[0081] In certain embodiments, a multispecific antibody, or a functional portion thereof, derivative, and / or analogue, or method by which the use of the present disclosure relates includes a variable domain that binds to the extracellular portion of PD-L1, wherein the antibody includes a heavy chain variable region that binds to the extracellular portion of PD-L1, comprising a CDR2 region having the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 94, SEQ ID NO: 101, or SEQ ID NO: 105, or a variant thereof.

[0082] In certain embodiments, a multispecific antibody, or a functional portion, derivative, and / or analog thereof, or method by use of the present disclosure comprising a variable domain that binds to the extracellular portion of PD-L1, includes a heavy chain variable region having a CDR1 region comprising the amino acid sequence of the CDR1 region of the variable heavy chain region of MF5554 (SEQ ID NO: 54), MF5576 (SEQ ID NO: 54), MF5578 (SEQ ID NO: 60), MF9375 (SEQ ID NO: 60), MF9376 (SEQ ID NO: 65), MF7702 (SEQ ID NO: 68), MF5424 (SEQ ID NO: 82), MF5561 (SEQ ID NO: 93), MF5439 (SEQ ID NO: 90), MF5553 (SEQ ID NO: 68), MF5594 (SEQ ID NO: 74), MF5426 (SEQ ID NO: 86), or MF5442 (SEQ ID NO: 93).

[0083] In certain embodiments, a multispecific antibody, or a functional portion thereof, derivative, and / or analogue, or method by use of the present disclosure comprising a variable domain that binds to the extracellular portion of PD-L1, includes a heavy chain variable region having a CDR1 region comprising the amino acid sequence of the CDR1 region of the variable heavy chain region of MF7702 (SEQ ID NO: 56), MF5424 (SEQ ID NO: 84), MF5561 (SEQ ID NO: 102), MF5439 (SEQ ID NO: 91), MF5553 (SEQ ID NO: 56), MF5594 (SEQ ID NO: 106), MF5426 (SEQ ID NO: 88), or MF5442 (SEQ ID NO: 95).

[0084] In certain embodiments, a multispecific antibody or method using the present disclosure comprising a variable domain that binds to the extracellular portion of PD-L1 includes a heavy chain variable region that binds to the extracellular portion of PD-L1, comprising an amino acid sequence represented by SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, or SEQ ID NO: 93, or a CDR1 region having a variant thereof.

[0085] In certain embodiments, a multispecific antibody, or a functional portion, derivative, and / or analog thereof, or a method by use of the present disclosure comprising a variable domain that binds to the extracellular portion of PD-L1, includes a heavy chain variable region having CDR1, CDR2, and CDR3 regions, which include the amino acid sequences of CDR1, CDR2, and CDR3 of one of the variable heavy chain regions of VH presented as MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5424, MF5561, MF5439, MF5553, MF5594, MF5426, or MF5442. The CDR1, CDR2, and CDR3 sequences are preferably selected from the same VH region.

[0086] In certain embodiments, the use of the Disclosure to obtain multispecific antibodies, or functional portions thereof, derivatives and / or analogs, or methods comprising a variable domain that binds to the extracellular portion of PD-L1, includes MF5554 (SEQ ID NO: 53), MF5576 (SEQ ID NO: 57), MF5578 (SEQ ID NO: 59), MF9375 (SEQ ID NO: 62), MF9376 (SEQ ID NO: 64), MF7702 (SEQ ID NO: 67), MF5424 (SEQ ID NO: 81), MF5561 (SEQ ID NO: 100), The variable heavy chain region amino acid sequence of MF5439 (SEQ ID NO: 89), MF5553 (SEQ ID NO: 96), MF5594 (SEQ ID NO: 104), MF5426 (SEQ ID NO: 85), or MF5442 (SEQ ID NO: 92) is included and has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 3, 4, 5, 6, 7, 8, 9, or 10, or 0, 1, 2, 3, 4, or 5, relative to the VH amino acid sequence of the indicated MF. In certain embodiments, the variable heavy chain region amino acid sequence has up to 5 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, 0, 1, or 2, or 0 or 1, relative to the VH amino acid sequence of the indicated MF. In certain embodiments, if present, amino acid insertions, deletions, substitutions, or combinations thereof are not present in the CDR region amino acid sequence.

[0087] In certain embodiments, particularly preferred combinations of multispecific antibodies, functional moieties, derivatives, and / or analogs, or methods using the present disclosure include MF6797 (SEQ ID NO: 49) and MF7702 (SEQ ID NO: 67), MF6763 (SEQ ID NO: 24) and MF7702 (SEQ ID NO: 67), MF6785 (SEQ ID NO: 31) and MF7702 (SEQ ID NO: 67), MF6797 (SEQ ID NO: 49) and MF5553 (SEQ ID NO: 96), MF6763 (SEQ ID NO: 24) and MF5553 (SEQ ID NO: 96), MF6785 (SEQ ID NO: 31) and The sequence is represented by MF5553 (sequence number 96), MF6754 (sequence number 20), and MF5424 (sequence number 81), MF6763 (sequence number 24), and MF5561 (sequence number 100), MF6785 (sequence number 31), and MF5439 (sequence number 89), MF6754 (sequence number 20), and MF5553 (sequence number 96), MF6744 (sequence number 28), and MF5594 (sequence number 104), or MF6783 (sequence number 1) and MF5594 (sequence number 104), or a combination of variable domains containing variants thereof.

[0088] In certain embodiments, the use of the multispecific antibodies, or their functional portions, derivatives, and / or analogs, or methods thereof, as described herein, - A CD137-binding variable domain comprising a VH region having the amino acid sequence of CDR3, or the amino acid sequences of CDR1, CDR2, and CDR3 of the VH region of MF6797 (SEQ ID NO: 49), -Includes the amino acid sequence of CDR3, or a PD-L1 binding variable domain containing a VH region having the amino acid sequences of CDR1, CDR2, and CDR3 of MF5554 (SEQ ID NO: 53), MF5576 (SEQ ID NO: 57), MF5578 (SEQ ID NO: 59), MF9375 (SEQ ID NO: 62), MF9376 (SEQ ID NO: 64), MF7702 (SEQ ID NO: 67), MF5594 (SEQ ID NO: 104), MF5424 (SEQ ID NO: 81), MF5426 (SEQ ID NO: 85), MF5553 (SEQ ID NO: 96), MF5442 (SEQ ID NO: 92), MF5561 (SEQ ID NO: 100), or MF5439 (SEQ ID NO: 89).

[0089] In certain embodiments, the use of the multispecific antibodies, or their functional portions, derivatives, and / or analogs, or methods thereof, as described herein, - A CD137-binding variable domain containing a VH region having the amino acid sequence of MF6797 (SEQ ID NO: 49), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of the VH region of MF6797 (SEQ ID NO: 49), -Includes a PD-L1 binding variable domain having a VH region containing the VH amino acid sequence of MF5554 (SEQ ID NO: 53), MF5576 (SEQ ID NO: 57), MF5578 (SEQ ID NO: 59), MF9375 (SEQ ID NO: 62), MF9376 (SEQ ID NO: 64), MF7702 (SEQ ID NO: 67), MF5594 (SEQ ID NO: 104), MF5424 (SEQ ID NO: 81), MF5426 (SEQ ID NO: 85), MF5553 (SEQ ID NO: 96), MF5442 (SEQ ID NO: 92), MF5561 (SEQ ID NO: 100), or MF5439 (SEQ ID NO: 89), which has 0, 1, 2, 3, 4, 5, or 10 amino acid insertions, deletions, substitutions, or combinations thereof with respect to the VH amino acid sequence of the indicated MF. In a particular embodiment, the amino acid sequence of the variable heavy chain region has up to five amino acid insertions, deletions, substitutions, or combinations thereof with respect to the amino acid sequence of the VH of the MF, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, 0, 1, or 2, or 0 or 1.

[0090] Certain embodiments of the Disclosure relate to multispecific antibodies, or functional portions thereof, derivatives, and / or analogs, or methods of use thereof. - A CD137-binding variable domain containing a VH region having the amino acid sequence of the CDR3 region of VH of MF6763 (SEQ ID NO: 27), The present invention further provides a multispecific antibody, or a functional portion thereof, derivatives, and / or analogue thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequence of the CDR3 of VH of -MF5442 (SEQ ID NO: 95).

[0091] A particular embodiment is a multispecific antibody, or a functional portion thereof, derivative, and / or analog thereof. -MF6763 (SEQ ID NO: 24) has a CD137-binding variable domain containing a VH region with amino acid sequences of the CDR1, CDR2, and CDR3 regions of the VH, The present invention provides a multispecific antibody, or a functional moiety, derivative, and / or analog thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the VH region of -MF5442 (SEQ ID NO: 92).

[0092] Certain embodiments of the Disclosure relate to multispecific antibodies, or functional portions thereof, derivatives, and / or analogs, or methods of use thereof. - A CD137-binding variable domain containing a VH region having the amino acid sequence of MF6763 (SEQ ID NO: 24), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of the VH of MF6763, The present invention provides a multispecific antibody, or a functional moiety, derivative, and / or analogue thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequence of MF5442 (SEQ ID NO: 92), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of VH of MF5442. In a particular embodiment, the amino acid sequence of the variable heavy chain region has up to 5 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, or 0, 1, or 2, or 0 or 1, relative to the amino acid sequence of VH of the indicated MF.

[0093] Certain embodiments of the Disclosure relate to multispecific antibodies, or functional portions thereof, derivatives, and / or analogs, or methods of use thereof. - A CD137-binding variable domain containing a VH region having the amino acid sequence of the CDR3 region of VH of MF6797 (SEQ ID NO: 52), The present invention further provides a multispecific antibody, or a functional moiety, derivative, and / or analog thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequence of the CDR3 of VH of -MF7702 (SEQ ID NO: 56).

[0094] A particular embodiment is a multispecific antibody, or a functional portion thereof, derivative, and / or analog thereof. -MF6797 (SEQ ID NO: 49) has a CD137-binding variable domain containing a VH region with amino acid sequences of the CDR1, CDR2, and CDR3 regions of the VH region, The present invention provides a multispecific antibody, or a functional moiety, derivative, and / or analog thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the VH region of -MF7702 (SEQ ID NO: 67).

[0095] Certain embodiments of the Disclosure relate to multispecific antibodies, or functional portions thereof, derivatives, and / or analogs, or methods of use thereof. - A CD137-binding variable domain containing a VH region having the amino acid sequence of MF6797 (SEQ ID NO: 49), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, or 10, or 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of the VH of MF6797, The present invention provides a multispecific antibody, or a functional moiety, derivative, and / or analogue thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequence of MF7702 (SEQ ID NO: 67), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of VH of MF7702. In a particular embodiment, the amino acid sequence of the variable heavy chain region has up to 5 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, or 0, 1, or 2, or 0 or 1, relative to the amino acid sequence of VH of the indicated MF.

[0096] Certain embodiments of the Disclosure relate to multispecific antibodies, or functional portions thereof, derivatives, and / or analogs, or methods of use thereof. - A CD137-binding variable domain containing a VH region having the amino acid sequence of the CDR3 region of VH of MF6754 (SEQ ID NO: 23), The present invention further provides a multispecific antibody, or a functional moiety, derivative, and / or analog thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequence of the CDR3 of VH of -MF5561 (SEQ ID NO: 102).

[0097] A particular embodiment is a multispecific antibody, or a functional portion thereof, derivative, and / or analog thereof. -MF6754 (SEQ ID NO: 20) has a CD137-binding variable domain containing a VH region with amino acid sequences for the CDR1, CDR2, and CDR3 regions of the VH region, The present invention provides a multispecific antibody, or a functional moiety, derivative, and / or analog thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the VH region of -MF5561 (SEQ ID NO: 100).

[0098] Certain embodiments of the Disclosure relate to multispecific antibodies, or functional portions thereof, derivatives, and / or analogs, or methods of use thereof. - A CD137-binding variable domain containing a VH region having the amino acid sequence of MF6754 (SEQ ID NO: 20), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, or 10, or 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of the VH of MF6754, The present invention provides a multispecific antibody, or a functional moiety, derivative, and / or analogue thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequence of MF5561 (SEQ ID NO: 100), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of VH of MF5561. In a particular embodiment, the amino acid sequence of the variable heavy chain region has up to 5 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, or 0, 1, or 2, or 0 or 1, relative to the amino acid sequence of VH of the indicated MF.

[0099] Certain embodiments of the Disclosure relate to multispecific antibodies, or functional portions thereof, derivatives, and / or analogs, or methods of use thereof. - A CD137-binding variable domain containing a VH region having the amino acid sequence of the CDR3 region of VH of MF6785 (SEQ ID NO: 34), The present invention further provides a multispecific antibody, or a functional portion thereof, derivatives, and / or analogue thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequence of the CDR3 of VH of -MF5439 (SEQ ID NO: 91).

[0100] Also provided are bispecific antibodies, or their functional portions, derivatives, and / or analogs. -MF6785 (SEQ ID NO: 31) has a CD137-binding variable domain containing a VH region with amino acid sequences for the CDR1, CDR2, and CDR3 regions of the VH region, A multispecific antibody, or a functional moiety, derivative, and / or analog thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the VH region of -MF5439 (SEQ ID NO: 89).

[0101] Certain embodiments of the Disclosure relate to multispecific antibodies, or functional portions thereof, derivatives, and / or analogs, or methods of use thereof. - A CD137-binding variable domain containing a VH region having the amino acid sequence of MF6785 (SEQ ID NO: 31), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of the VH of MF6785, The present invention provides a multispecific antibody, or a functional moiety, derivative, and / or analogue thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequence of MF5439 (SEQ ID NO: 89), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of VH of MF5439. In a particular embodiment, the amino acid sequence of the variable heavy chain region has up to 5 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, or 0, 1, or 2, or 0 or 1, relative to the amino acid sequence of VH of the indicated MF.

[0102] Certain embodiments of the Disclosure relate to multispecific antibodies, or functional portions thereof, derivatives, and / or analogs, or methods of use thereof. - A CD137-binding variable domain containing a VH region having the amino acid sequence of the CDR3 region of VH of MF6785 (SEQ ID NO: 34), The present invention further provides a multispecific antibody, or a functional portion thereof, derivatives, and / or analogue thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequence of the CDR3 of VH of -MF5542 (SEQ ID NO: 95).

[0103] A particular embodiment is a multispecific antibody, or a functional portion thereof, derivative, and / or analog thereof. -MF6785 (SEQ ID NO: 31) has a CD137-binding variable domain containing a VH region with amino acid sequences for the CDR1, CDR2, and CDR3 regions of the VH region, The present invention provides a multispecific antibody, or a functional moiety, derivative, and / or analog thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the VH region of -MF5442 (SEQ ID NO: 92).

[0104] Certain embodiments of the Disclosure relate to multispecific antibodies, or functional portions thereof, derivatives, and / or analogs, or methods of use thereof. - A CD137-binding variable domain containing a VH region having the amino acid sequence of MF6785 (SEQ ID NO: 31), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of the VH of MF6785, The present invention provides a multispecific antibody, or a functional moiety, derivative, and / or analogue thereof, comprising a PD-L1 binding variable domain containing a VH region having the amino acid sequence of MF5442 (SEQ ID NO: 92), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 0, 1, 2, 3, 4, or 5, relative to the amino acid sequence of VH of MF5542. In a particular embodiment, the amino acid sequence of the variable heavy chain region has up to 5 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, or 0, 1, or 2, or 0 or 1, relative to the amino acid sequence of VH of the indicated MF.

[0105] Certain embodiments of the use of the Disclosure include multispecific antibodies, or functional portions thereof, derivatives, and / or analogs, or methods thereof. -Includes CDR1 according to SEQ ID NO: 50, CDR2 according to SEQ ID NO: 51, and CDR3 according to SEQ ID NO: 52, where each CDR1, CDR2, and / or CDR3 is a variable domain having 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, or -Includes CDR1 as described in SEQ ID NO: 40, CDR2 as described in SEQ ID NO: 41, and CDR3 as described in SEQ ID NO: 42, where each CDR1, CDR2, and / or CDR3 is a variable domain having 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, or -Includes CDR1 according to SEQ ID NO: 21, CDR2 according to SEQ ID NO: 22, and CDR3 according to SEQ ID NO: 23, where each CDR1, CDR2, and / or CDR3 is a variable domain having 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, or - A binding domain that binds to CD137, comprising CDR1 according to SEQ ID NO: 32, CDR2 according to SEQ ID NO: 33, and CDR3 according to SEQ ID NO: 34, wherein each CDR1, CDR2, and / or CDR3 contains a variable domain having 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, and / or -Includes CDR1 according to SEQ ID NO: 68, CDR2 according to SEQ ID NO: 55, and CDR3 according to SEQ ID NO: 56, where each CDR1, CDR2, and / or CDR3 is a variable domain having 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, or -Includes CDR1 according to SEQ ID NO: 93, CDR2 according to SEQ ID NO: 94, and CDR3 according to SEQ ID NO: 95, where each CDR1, CDR2, and / or CDR3 is a variable domain having 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, or -Includes CDR1 according to SEQ ID NO: 93, CDR2 according to SEQ ID NO: 101, and CDR3 according to SEQ ID NO: 102, where each CDR1, CDR2, and / or CDR3 is a variable domain having 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, or The present invention provides a multispecific antibody, or a functional moiety, derivative, and / or analog thereof, comprising CDR1 according to SEQ ID NO: 90, CDR2 according to SEQ ID NO: 79, and CDR3 according to SEQ ID NO: 91, wherein each CDR1, CDR2, and / or CDR3 comprises a binding domain that binds to PD-L1, each containing a variable domain having 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof. In a particular embodiment, the amino acid sequence of the CDR has up to 5 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, 0, 1, or 2, or 0 or 1.

[0106] In certain embodiments, a multispecific antibody, or a functional portion, derivative, and / or analog thereof, or a method of use of the Disclosure, includes a variable domain that binds to the extracellular portion of CD137, thereby blocking the binding of CD137 to its ligand, and a variable domain that binds to the extracellular portion of PD-L1, thereby blocking the binding of PD-1 to PD-L1. In certain embodiments, the variable domain that binds to the extracellular portion of PD-L1 in the antibody, or a functional portion, derivative, and / or analog thereof, includes the amino acid sequence of CDR3, or a VH region having the amino acid sequence of CDR1, CDR2, and CDR3 of one of the VH regions of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5424, MF5561, MF5439, MF5553, MF5594, MF5426, and MF5442. In a particular embodiment, the variable domain that binds to the extracellular portion of PD-L1 includes a VH region having the VH amino acid sequence of MF5554 (SEQ ID NO: 53), MF5576 (SEQ ID NO: 57), MF5578 (SEQ ID NO: 59), MF9375 (SEQ ID NO: 62), MF9376 (SEQ ID NO: 64), MF7702 (SEQ ID NO: 67), MF5424 (SEQ ID NO: 81), MF5561 (SEQ ID NO: 100), MF5439 (SEQ ID NO: 89), MF5553 (SEQ ID NO: 96), MF5594 (SEQ ID NO: 104), MF5426 (SEQ ID NO: 85), MF5442 (SEQ ID NO: 92), which has up to 15 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, 4, 5, or 10, or 0, 1, 2, 3, 4, or 5, relative to the VH amino acid sequence of the indicated MF. In a particular embodiment, the amino acid sequence of the variable heavy chain region has up to five amino acid insertions, deletions, substitutions, or combinations thereof with respect to the amino acid sequence of the VH of the MF, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, 0, 1, or 2, or 0 or 1.

[0107] This is demonstrated in the example of WO2018 / 056821A1, where the binding of CD137-specific VH to MF6797 (SEQ ID NO: 49) is associated with the presence of amino acids including Arg66, Gly70, and Phe72 in the CD137 amino acid sequence.

[0108] Accordingly, in certain embodiments, the Disclosure also provides the use of or therapeutic methods of isolated, synthesized, or recombinant antibodies, or functional moieties, derivatives, and / or analogs thereof that are capable of binding to CD137, the binding of such antibodies, functional moieties, derivatives, or analogs to CD137 is associated with the presence of amino acids including Arg66, Gly70, and Phe72 in the CD137 amino acid sequence (SEQ ID NO: 117). In certain embodiments, the binding of such antibodies, functional moieties, derivatives, or analogs to CD137 is also associated with amino acids including Val71 in the CD137 amino acid sequence.

[0109] The term "Arg66" refers to the arginine residue at position 66 of the CD137 sequence. The term "Gly70" refers to the glycine residue at position 70 of the CD137 sequence according to SEQ ID NO: 117. The term "Val71" refers to the valine residue at position 71 of the CD137 sequence. The term "Phe72" refers to the phenylalanine residue at position 72 of the CD137 sequence.

[0110] In certain embodiments, the multispecific antibodies of the present disclosure include binding sites comprising a common light chain according to SEQ ID NO: 109, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. In certain embodiments, each binding site comprises a common light chain according to SEQ ID NO: 109, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof.

[0111] In certain embodiments, the multispecific antibodies of the present disclosure include binding sites comprising an IMGT-based LCDR sequence of the common light chain according to SEQ ID NO: 109, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. In certain embodiments, each binding site comprises an IMGT-based LCDR sequence of the common light chain according to SEQ ID NO: 109, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. In certain embodiments, the substitutions are conservative substitutions in which an amino acid is replaced by a conserved amino acid. Those skilled in the art will be well able to substitute one or more amino acids in the CDR sequence according to the present disclosure. For example, conservative amino acid substitutions are applied. Examples of conservative amino acid substitutions include the substitution of one hydrophobic residue, e.g., isoleucine, valine, leucine, or methionine, for another hydrophobic residue, and the substitution of one polar residue for another polar residue, e.g., arginine to lysine, glutamic acid to aspartic acid, or glutamine to asparagine. In certain embodiments, the multispecific antibodies of this disclosure include a binding site comprising an LCDR sequence (according to the IMGT database World Wide Web at imgt.org) of the common light chain according to SEQ ID NO: 109, having one or two amino acid insertions, deletions, substitutions, additions, or combinations thereof. In certain embodiments, each binding site comprises an LCDR sequence (according to IMGT) of the common light chain according to SEQ ID NO: 109, having one or two amino acid insertions, deletions, substitutions, additions, or combinations thereof.

[0112] In certain embodiments, the light chain variable regions of one or more binding domains of the multispecific antibodies of the present disclosure include LCDR1 containing the amino acid sequence QSISSY, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence QQSYSTPPT (i.e., the CDRs of IGKV1-39 by IMGT).

[0113] In certain embodiments, one or more binding domains of the multispecific antibody of the present disclosure include a light chain variable region containing an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 109 in certain embodiments.

[0114] In certain embodiments, the multispecific antibodies described herein include a common light chain variable domain, such as the common light chain variable region of SEQ ID NO: 110, having 0, 1, 2, 3, or 4 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, or 4, or 0, 1, 2, or 3, or 0, 1, or 2, or 0 or 1 amino acid insertions, deletions, substitutions, or combinations thereof. In certain embodiments, the multispecific antibodies described herein include a common light chain constant domain, such as the common light chain constant region of SEQ ID NO: 111, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. The term "common light chain" in the present invention refers to a light chain that may be identical or may have some differences in amino acid sequences, but whose binding specificity of the full-length antibody is not affected. For example, it is possible, within the scope of the definition of a common light chain as used herein, to prepare or find a light chain that is not identical but is still functionally equivalent by introducing and testing conservative amino acid changes, or changes in amino acids in regions that do not contribute to binding specificity, or only partially contribute, when paired with a heavy chain. The terms “common light chain,” “common LC,” “cLC,” and “single light chain” are all used interchangeably herein, with or without the addition of the term “rearranged.” The terms “common light chain variable region,” “common VL,” “common LCv,” “cLCv,” and “single VL” are all used interchangeably herein, with or without the addition of the term “rearranged.” In certain aspects of this disclosure, a multispecific antibody has a common light chain (variable region) that can be combined with at least two or more heavy chains (variable regions) of different binding specificities to form an antibody having a functional antigen-binding domain (WO2004 / 009618, WO2009 / 157771). In certain embodiments, the common light chain (variable region) is a human light chain (variable region). In certain embodiments, the common light chain (variable region) has a germline sequence. In certain embodiments, the germline sequence is a light chain variable region that is frequently used in the human repertoire and has good thermodynamic stability, yield, and solubility.In certain embodiments, the common light chain is a rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01 (SEQ ID NO: 109). In certain embodiments, the common light chain variable region is the variable region of the rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01. In certain embodiments, the common light chain includes a light chain variable region presented in SEQ ID NO: 110, having 0 to 5 amino acid insertions, deletions, substitutions, or combinations thereof, such as 0, 1, 2, 3, or 4, 0, 1, 2, or 3, 0, 1, or 2, or 0 or 1. In certain embodiments, the common light chain further includes a light chain constant region, such as a kappa light chain constant region. The nucleic acid encoding the common light chain can be codon-optimized for the cell line used to express the common light chain protein. The coding nucleic acid can deviate from the germline nucleic acid sequence.

[0115] In certain embodiments, the multispecific antibody or method used in this disclosure is a full-length antibody or an antibody fragment, such as a Fab fragment or a single-stranded variable fragment (scFv). In certain embodiments, the multispecific antibody or method used in this disclosure is a full-length antibody.

[0116] In certain embodiments, the multispecific antibody or method used in this disclosure is IgG. In certain embodiments, the multispecific antibody is an IgG1 molecule that does not have Fc effector function.

[0117] The Fc region mediates the effector functions of antibodies, such as complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and antibody-dependent phagocytosis (ADCP). Depending on the application of the therapeutic antibody or Fc fusion protein, it may be desirable to reduce or increase the effector function. Reduced effector function is preferred in this disclosure. Reduced effector function may be desired when an immune response is activated, enhanced, or stimulated, as in some aspects of this disclosure. Antibodies with reduced effector function can be used to target, among other things, cell surface molecules of immune cells. In certain aspects, the CH2 region of the multispecific antibody of this disclosure is engineered to reduce the ADCC and / or CDC activity of the antibody. In certain aspects, the CH3 region of the multispecific antibody is engineered to promote heavy chain heterodimerization.

[0118] Antibodies with reduced effector function are, for example, IgG antibodies that include a modified CH2 / lower hinge region to reduce Fc receptor interaction or C1q binding. In certain embodiments, the antibody of the Disclosure is an IgG antibody having a mutant CH2 and / or lower hinge domain such that the interaction of the multispecific IgG antibody with the Fc-gamma receptor is reduced. In certain embodiments, the CH2 region of the Disclosure includes the amino acid sequence described in SEQ ID NO: 114, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. In certain embodiments, the hinge region of the Disclosure includes the amino acid sequence described in SEQ ID NO: 113, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof.

[0119] In certain embodiments, the CH3 region of a multispecific antibody is manipulated to promote heterodimerization of the heavy chain. In certain embodiments, these variations exist solely to produce multispecific full-length IgG molecules having amino acid substitutions at positions 351 and 366 in the first CH3 domain ("KK variant" heavy chain), e.g., L351K and T366K (numbered according to EU numbering), and amino acid substitutions at positions 351 and 368 in the second CH3 domain ("DE variant" heavy chain), e.g., L351D and L368E, or vice versa. Homodimerization of the DE variant heavy chain (DE-DE homodimer) or the KK variant heavy chain (KK-KK homodimer) is rarely observed due to strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains. In certain embodiments, the multispecific antibodies of the present disclosure include the CH3 domains of SEQ ID NOs: 115 and 116, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof, provided that the DE / KK variant is not altered.

[0120] In certain embodiments, PD-1 or PD-L1 inhibitors are antibodies or other binding molecules.

[0121] In certain embodiments, the PD-1 or PD-L1 inhibitor is a PD-1 inhibitor. In certain embodiments, the PD-1 inhibitor is selected from nivolumab, pembrolizumab, semiprimab, penprimab, retifanlimab, cintilimab, tislerizumab, tripalimab, and dostarimab. In certain embodiments, the PD-1 inhibitor is pembrolizumab. In certain embodiments, the PD-1 or PD-L1 inhibitor is a PD-L1 inhibitor. In certain embodiments, the PD-L1 inhibitor is selected from atezolizumab, avelumab, and durvalumab. The amino acid sequences of nivolumab, pembrolizumab, semiprimab, dostarimab, atezolizumab, avelumab, and durvalumab are well known in the art.

[0122] In certain embodiments, PD-1 or PD-L1 inhibitors are administered in accordance with these recommendations established by FDA approval.

[0123] In a particular embodiment, the PD-1 inhibitor is nivolumab, administered at a dose of 3 mg / kg as an intravenous infusion (e.g., over 60 minutes) every two weeks until disease progression or unacceptable toxicity.

[0124] In an alternative configuration, nivolumab is administered at a dose of 240 mg every two weeks, or 480 mg every four weeks, or 1 mg / kg every three weeks on the same day, followed by 3 mg / kg of ipilimumab four times, then 240 mg every two weeks, or 480 mg every four weeks.

[0125] In an alternative embodiment, nivolumab is administered every 6 weeks at a dose of 3 mg / kg every 2 weeks with ipilimumab 1 mg / kg, or every 6 weeks at a dose of 360 mg every 3 weeks with ipilimumab 1 mg / kg, and in two cycles of platinum-doublet chemotherapy.

[0126] In an alternative configuration, nivolumab is administered every three weeks at a dose of 360 mg, along with ipilimumab at a dose of 1 mg / kg every six weeks.

[0127] In an alternative embodiment, nivolumab is administered at 240 mg every two weeks, or 480 mg every four weeks, or 3 mg / kg every two weeks, or 3 mg / kg on the same day every three weeks, followed by ipilimumab 1 mg / kg four times, then 240 mg every two weeks, or 480 mg every four weeks.

[0128] In a particular embodiment, the PD-1 inhibitor is cemiprimab, administered at a dose of 350 mg every three weeks (e.g., over 30 minutes) as an intravenous infusion.

[0129] In a particular embodiment, the PD-1 inhibitor is dostalimab, administered as an intravenous infusion (e.g., over 30 minutes) starting with a first to fourth dose of 500 mg every three weeks, followed by subsequent doses starting three weeks after dose 4 (dose 5 and beyond): 1,000 mg every six weeks, until disease progression or unacceptable toxicity occurs.

[0130] In a particular embodiment, the PD-L1 inhibitor is atezolizumab, administered at a dose of 1200 mg every three weeks (e.g., over 60 minutes) as an intravenous infusion. If the initial infusion is tolerable, all subsequent infusions may be delivered over 30 minutes.

[0131] In a particular embodiment, the PD-L1 inhibitor is avelumab, administered at a dose of 10 mg / kg every two weeks (e.g., over 60 minutes) as an intravenous infusion.

[0132] In a particular embodiment, the PD-L1 inhibitor is durvalumab, administered at a dose of 10 mg / kg as an intravenous infusion (e.g., over 60 minutes) every two weeks until disease progression or unacceptable toxicity.

[0133] In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion of 400 mg or as an intravenous infusion of 2 mg / kg every three weeks. In certain embodiments, administration is a uniform dose of 400 mg every six weeks until disease progression or unacceptable toxicity.

[0134] In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion at a dose of 400 mg every six weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered as a uniform dose of 400 mg every six weeks as an intravenous infusion. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion at a dose of 200 mg every three weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered as a uniform dose of 200 mg every three weeks as an intravenous infusion. In certain embodiments, administration is a uniform dose of 400 mg every six weeks until disease progression or unacceptable toxicity. In certain embodiments, administration is a uniform dose of 200 mg every three weeks until disease progression or unacceptable toxicity.

[0135] In this specification, the term “uniform dose” refers to a dosing regimen in which a subject is administered a fixed amount of therapeutic agent over multiple doses, regardless of the subject’s body weight. Uniform dosing is typically abbreviated as qnw, where n is an integer representing the interval and w is a week. For example, a q6w uniform dose dosing regimen of 400 mg antibody means that a fixed amount of 400 mg of antibody is administered each for 6 weeks. In certain embodiments, pembrolizumab is administered in a 400 mg q6w dosing regimen. In certain embodiments, pembrolizumab is administered in a 200 mg q3w dosing regimen.

[0136] A uniform dose may be premedicated, meaning that a drug is administered to the subject before the administration of the antibody of this disclosure. In certain embodiments, a uniform dose of pembrolizumab or the multispecific antibody of this disclosure may be premedicated with an antihistamine, analgesic, antipyretic, and / or anti-inflammatory drug.

[0137] In certain embodiments, the PD-1 or PD-L1 inhibitor is a full-length antibody or a fragment of an antibody, such as a Fab fragment or a single-stranded variable fragment (scFv). In certain embodiments, the PD-1 or PD-L1 inhibitor or method used by the Disclosure is a full-length antibody. In certain embodiments, the PD-1 or PD-L1 inhibitor is an antibody selected from nivolumab, pembrolizumab, semiprimab, dostarimab, atezolizumab, avelumab, and durvalumab, or a functional fragment or variant thereof, or includes them.

[0138] In certain embodiments, the PD-1 or PD-L1 inhibitor is the amino acid sequence of the antigen-binding site of one of nivolumab, pembrolizumab, semiprimab, dostarimab, atezolizumab, avelumab, and durvalumab, particularly pembrolizumab, or comprises or consists of an amino acid sequence having substantial sequence identity with them.

[0139] In certain embodiments, the PD-1 or PD-L1 inhibitor is one of nivolumab, pembrolizumab, semiprimab, dostarimab, atezolizumab, avelumab, and durvalumab, and includes at least one, at least two, and more than three CDRs that are identical or substantially identical to the CDR of pembrolizumab. In certain embodiments, the PD-1 or PD-L1 inhibitor includes at least one, at least two, and more than three CDRs that are identical to the CDRs found in pembrolizumab.

[0140] In certain embodiments, the PD-1 or PD-L1 inhibitor is CDR1, which is identical to CDR1 from one of nivolumab, pembrolizumab, semiprimab, dostarimab, atezolizumab, avelumab, and durvalumab; CDR2, which is identical to CDR2 from one of nivolumab, pembrolizumab, semiprimab, dostarimab, atezolizumab, avelumab, and durvalumab; or CDR3, which is identical to CDR3 from one of nivolumab, pembrolizumab, semiprimab, dostarimab, atezolizumab, avelumab, and durvalumab. In certain embodiments, the PD-1 or PD-L1 inhibitor is CDR1, which is identical to CDR1 from one of nivolumab, pembrolizumab, semiprimab, dostarimab, atezolizumab, avelumab, and durvalumab. CDR1 and CDR2 that are identical to CDR1 and CDR2 from one of the following, CDR1 and CDR3 that are identical to CDR1 and CDR3 from one of the following, nivolumab, pembrolizumab, semiprimab, dostarimab, atezolizumab, avelumab, and durvalumab, or CDR2 and CDR3 that are identical to CDR2 and CDR3 from one of the following, nivolumab, pembrolizumab, semiprimab, dostarimab, atezolizumab, avelumab, and durvalumab, further including CDR1, CDR2, and CDR3 that are identical to CDR1, CDR2, and CDR3 of nivolumab, pembrolizumab, semiprimab, dostarimab, atezolizumab, avelumab, and durvalumab, etc. In certain embodiments, the PD-1 or PD-L1 inhibitor includes CDR1, CDR2, and CDR3, which are identical to CDR1, CDR2, and CDR3 derived from pembrolizumab.

[0141] In certain embodiments, the multispecific antibody is administered in doses of 10 to 300 mg, such as 10 to 150 mg or 25 to 100 mg, or 25 to 75 mg. In certain embodiments, the multispecific antibody is administered in doses of 25, 30, 35, 40, 45, or 50 mg. In certain embodiments, the multispecific antibody is administered in uniform doses of 25, 30, 35, 40, 45, or 50 mg.

[0142] In a particular embodiment, the multispecific antibody is administered approximately every 7, 10, 11, 12, 13, 14, 15, 16, 17, or 18 or 21 days, particularly every 10, 14, or 18 days, and more specifically every 14 days.

[0143] In certain embodiments, PD-1 or PD-L1 inhibitors are administered in doses and schedules approved by the USFDA. In certain embodiments, pembrolizumab is administered in doses of approximately 300–500 mg, such as 375–425 mg, such as 350–450 mg. In certain embodiments, PD-1 or PD-L1 inhibitors are administered in doses of 325, 350, 375, 400, 425, 450, or 475 mg.

[0144] In some embodiments, the multispecific antibody is administered in doses of 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 75 mg, 100 mg, 125 mg, or 150 mg.

[0145] In certain embodiments, the multispecific antibodies or methods used in this disclosure are administered in a uniform dose.

[0146] In certain embodiments, the multispecific antibody or method used in this disclosure is administered in doses of 50-150 mg, such as 75-150 mg, 25-125 mg, or 100-150 mg, or a uniform dose. In certain embodiments, the multispecific antibody or method used in this disclosure is administered in doses of 50-150 mg, 75-150 mg, 75-125 mg, or 100-150 mg, or a uniform dose.

[0147] In certain embodiments, the multispecific antibody or method used in this disclosure is administered in a dose of 75 to 125 mg or a uniform dose.

[0148] In certain embodiments, the multispecific antibody or method used in this disclosure is administered in a dose of 50-150 mg, such as 75-125 mg, or a uniform dose. In certain embodiments, the multispecific antibody or method used in this disclosure is administered in a dose of 50-100 mg, or 75-100 mg, or 75-125 mg, or a uniform dose.

[0149] In certain embodiments, the multispecific antibody or method used in this disclosure is administered in a dose of 25 to 75 mg or a uniform dose.

[0150] In certain embodiments, the multispecific antibody or method used in this disclosure is administered in a dose of 10-50 mg, such as 25-50 mg, or in a uniform dose.

[0151] In certain embodiments, the multispecific antibody or method used in this disclosure is administered once weekly, once every two weeks, or once every three weeks. In certain embodiments, the multispecific antibody or method used in this disclosure is administered once every two weeks. In certain embodiments, when the multispecific antibody or method used in this disclosure is administered once weekly, the multispecific antibody is administered in doses of 10 to 100 mg, such as 15 to 75 mg. In certain embodiments, when the multispecific antibody or method used in this disclosure is administered once weekly, the multispecific antibody is administered in doses of 10 to 100 mg, or 15 to 75 mg, such as 15 to 50 mg, 15 to 40 mg, 15 to 30 mg, or 15 to 25 mg.

[0152] In certain embodiments, the multispecific antibody or method used in this disclosure is administered in a uniform dose once every two weeks.

[0153] In certain embodiments, the multispecific antibody or method used in this disclosure is administered once every two weeks in a dose of 25, 30, 35, 40, 45, or 50 mg or a uniform dose. In certain embodiments, the multispecific antibody or method used in this disclosure is administered once every two weeks in a dose of 25 mg or a uniform dose. In certain embodiments, the multispecific antibody or method used in this disclosure is administered once every two weeks in a dose of 40 mg or a uniform dose. In certain embodiments, the multispecific antibody or method used in this disclosure is administered once every two weeks in a dose of 50 mg or a uniform dose.

[0154] In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in an intravenous infusion of 200 mg every three weeks, and the treatment further comprises administering the multispecific antibody of the Disclosure in an infusion of 10 mg every two weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in an intravenous infusion of 200 mg every three weeks, and the treatment further comprises administering the multispecific antibody of the Disclosure in an infusion of 15 mg every two weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in an intravenous infusion of 200 mg every three weeks, and the treatment further comprises administering the multispecific antibody of the Disclosure in an infusion of 25 mg every two weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in an intravenous infusion of 200 mg every three weeks, and the treatment further comprises administering the multispecific antibody of the Disclosure in an infusion of 40 mg every two weeks.

[0155] In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 400 mg intravenously every six weeks, and the treatment further includes administering the multispecific antibody of the Disclosure in a dose of 10 mg every two weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 400 mg intravenously every six weeks, and the treatment further includes administering the multispecific antibody of the Disclosure in a dose of 15 mg every three weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 400 mg intravenously every six weeks, and the treatment further includes administering the multispecific antibody of the Disclosure in a dose of 25 mg every two weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 400 mg intravenously every six weeks, and the treatment further includes administering the multispecific antibody of the Disclosure in a dose of 40 mg every two weeks.

[0156] In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 200 mg intravenously every three weeks, and the treatment further includes administering the multispecific antibody of the Disclosure in a dose of 10 mg once every three weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 200 mg intravenously every three weeks, and the treatment further includes administering the multispecific antibody of the Disclosure in a dose of 15 mg once every three weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 200 mg intravenously every three weeks, and the treatment further includes administering the multispecific antibody of the Disclosure in a dose of 25 mg once every three weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 200 mg intravenously every three weeks, and the treatment further includes administering the multispecific antibody of the Disclosure in a dose of 40 mg once every three weeks.

[0157] In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 400 mg intravenously every six weeks, and the treatment further comprises administering the multispecific antibody of the Disclosure in a dose of 10 mg every three weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 400 mg intravenously every six weeks, and the treatment further comprises administering the multispecific antibody of the Disclosure in a dose of 15 mg every three weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 400 mg intravenously every six weeks, and the treatment further comprises administering the multispecific antibody of the Disclosure in a dose of 25 mg every three weeks. In certain embodiments, the PD-1 inhibitor is pembrolizumab, administered in a dose of 400 mg intravenously every six weeks, and the treatment further comprises administering the multispecific antibody of the Disclosure in a dose of 40 mg every three weeks.

[0158] In certain embodiments, the multispecific antibodies or methods used in this disclosure are administered intravenously.

[0159] In certain embodiments, the multispecific antibodies or methods used in this disclosure are administered over a period of 30 minutes to 4 hours, such as 1 to 3 hours or 2 hours.

[0160] In certain embodiments, the multispecific antibodies or methods used in this disclosure are administered over a period of 30 minutes to 4 hours, such as 1 to 3 hours or 2 hours.

[0161] In certain embodiments, the multispecific antibodies or methods used in the present disclosure are administered intravenously in a uniform dose over a 2-hour period every 14 days in a 28-day cycle.

[0162] In certain embodiments, the multispecific antibody or method used in this disclosure is administered intravenously as a (uniform) dose of 10 mg over a 2-hour period every 14 days in a 28-day cycle. In certain embodiments, the multispecific antibody or method used in this disclosure is administered intravenously as a (uniform) dose of 15 mg over a 2-hour period every 14 days in a 28-day cycle. In certain embodiments, the multispecific antibody or method used in this disclosure is administered intravenously as a (uniform) dose of 25 mg over a 2-hour period every 14 days in a 28-day cycle. In certain embodiments, the multispecific antibody or method used in this disclosure is administered intravenously as a (uniform) dose of 30 mg over a 2-hour period every 14 days in a 28-day cycle. In certain embodiments, the multispecific antibody or method used in this disclosure is administered intravenously as a (uniform) dose of 40 mg over a 2-hour period every 14 days in a 28-day cycle. In certain embodiments, the multispecific antibody or method used in this disclosure is administered intravenously as a (uniform) dose of 50 mg over a 2-hour period every 14 days in a 28-day cycle. In certain embodiments, the multispecific antibody or method used in this disclosure is administered intravenously as a (uniform) dose of 60 mg over a 2-hour period every 14 days in a 28-day cycle. In certain embodiments, the multispecific antibody or method used in this disclosure is administered intravenously as a (uniform) dose of 70 mg over a 2-hour period every 14 days in a 28-day cycle. In certain embodiments, the multispecific antibody or method used in this disclosure is administered intravenously as a (uniform) dose of 75 mg over a 2-hour period every 14 days in a 28-day cycle.

[0163] In certain embodiments, the multispecific antibody or method used in this disclosure is formulated as a liquid at a concentration of 1 mg / mL to 100 mg / mL, such as 20 mg / mL or approximately 20 mg / mL. In certain embodiments, the multispecific antibody or method used in this disclosure is formulated as a liquid at a concentration of 1 mg / mL to 100 mg / mL, or 20 mg / mL or approximately 20 mg / mL.

[0164] In certain embodiments, pembrolizumab is administered in doses of 100-500 mg, or 100-300 mg, or 300-500 mg, or 150-250 mg, or 350-450 mg, or 175-225 mg, or 375-425 mg, or a uniform dose.

[0165] In certain embodiments, PD-1 or PD-L1 inhibitors are administered in doses of approximately 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, or 475 mg, or in doses of approximately 175 mg, 200 mg, 225 mg, 375 mg, 400 mg, or 425 mg, or in uniform doses.

[0166] In certain embodiments, PD-1 or PD-L1 inhibitors such as pembrolizumab are administered once every three weeks. In certain embodiments, PD-1 or PD-L1 inhibitors such as pembrolizumab are administered once every six weeks.

[0167] In certain embodiments, pembrolizumab is administered every three weeks in doses of 100-300 mg, 150-250 mg, 175-225 mg, or a uniform dose. In certain embodiments, pembrolizumab is administered every six weeks in doses of 300-500 mg, 350-450 mg, 375-425 mg, or a uniform dose.

[0168] In certain embodiments, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered every 21 days in doses of approximately 150 mg, 175 mg, 200 mg, 225 mg, or 250 mg, or a uniform dose. In certain embodiments, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered every 42 days in doses of approximately 350 mg, 375 mg, 400 mg, 425 mg, or 450 mg, or a uniform dose.

[0169] In certain embodiments, the PD inhibitor is first administered approximately 21 days after the first dose of the multispecific antibody. In certain embodiments, the PD inhibitor is first administered approximately 42 days after the first dose of the multispecific antibody.

[0170] In certain embodiments, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered in doses of approximately 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, or a uniform dose every 21 days. In certain embodiments, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered in doses of approximately 350 mg, 375 mg, 400 mg, 425 mg, or 450 mg, or a uniform dose, every 42 days. In certain embodiments, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered in doses of approximately 200 mg, or a uniform dose, every 21 days. In certain embodiments, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered in doses of approximately 400 mg, or a uniform dose, every 42 days.

[0171] In certain embodiments, PD-1 or PD-L1 inhibitors are administered intravenously, particularly via intravenous infusion. Such infusions may be administered over, for example, 15, 30, or 45 minutes.

[0172] In certain embodiments, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered via IV infusion every 21 days in doses of approximately 150 mg, 175 mg, 200 mg, 225 mg, or 250 mg, or a uniform dose. In certain embodiments, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered via IV infusion every 42 days in doses of approximately 350 mg, 375 mg, 400 mg, 425 mg, or 450 mg, or a uniform dose.

[0173] In certain embodiments, the multispecific antibody is administered every 14 days in doses of 10 mg, 25 mg, 30 mg, 40 mg, or 50 mg, or a uniform dose, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered every 21 days in doses of approximately 150 mg, 175 mg, 200 mg, 225 mg, or 250 mg, or a uniform dose. In certain embodiments, the multispecific antibody is administered every 14 days in doses of 10 mg, 25 mg, 30 mg, 40 mg, or 50 mg, or a uniform dose, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered every 42 days in doses of approximately 350 mg, 375 mg, 400 mg, 425 mg, or 450 mg, or a uniform dose.

[0174] In a particular embodiment, the multispecific antibody is administered in a uniform dose of 10 mg, 15 mg, 25 mg, or 40 mg every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered in a uniform dose of approximately 200 mg every 21 days. In a particular embodiment, the multispecific antibody is administered in a uniform dose of 10 mg, 15 mg, 25 mg, or 40 mg every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered in a uniform dose of approximately 400 mg every 42 days.

[0175] In a particular embodiment, the multispecific antibody is administered in a uniform dose of 10 mg, 15 mg, 25 mg, or 40 mg every 21 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered in a uniform dose of approximately 200 mg every 21 days. In a particular embodiment, the multispecific antibody is administered in a uniform dose of 10 mg, 15 mg, 25 mg, or 40 mg every 21 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered in a uniform dose of approximately 400 mg every 42 days.

[0176] In a particular embodiment, the multispecific antibody is administered at a dose of 40 mg or a uniform dose every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of approximately 200 mg or a uniform dose every 21 days, or at a dose of approximately 400 mg or a uniform dose every 42 days.

[0177] In a particular embodiment, the multispecific antibody is administered at a dose of 40 mg or a uniform dose every 21 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of approximately 200 mg or a uniform dose every 21 days, or at a dose of approximately 400 mg or a uniform dose every 42 days.

[0178] In a particular embodiment, the multispecific antibody is administered at a dose of 25 mg or a uniform dose every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of approximately 200 mg or a uniform dose every 21 days, or at a dose of approximately 400 mg or a uniform dose every 42 days.

[0179] In a particular embodiment, the multispecific antibody is administered at a dose of 30 mg or a uniform dose every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of approximately 200 mg or a uniform dose every 21 days, or at a dose of approximately 400 mg or a uniform dose every 42 days.

[0180] In a particular embodiment, the multispecific antibody is administered at a dose of 40 mg or a uniform dose every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of approximately 200 mg or a uniform dose every 21 days, or at a dose of approximately 400 mg or a uniform dose every 42 days.

[0181] In a particular embodiment, the multispecific antibody is administered at a dose of 50 mg or a uniform dose every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of approximately 200 mg or a uniform dose every 21 days, or at a dose of approximately 400 mg or a uniform dose every 42 days.

[0182] In certain embodiments, PD-1 or PD-L1 inhibitors are formulated as liquids with concentrations of 1 mg / mL to 100 mg / mL, or 25 mg / mL or around 25 mg / mL.

[0183] In certain embodiments, the multispecific antibodies or methods used in this disclosure are administered before, simultaneously with, or after antihistamines, nonsteroidal anti-inflammatory drugs (NSAIDs), narcotics, intravenous fluids, antipyretics, bronchodilators, oxygen, corticosteroids (IV / oral), vasoconstrictors, or any combination thereof, to reduce infusion-related reactions. If infusion-related reactions occur, premedication using the indicated substances may be selected for the subject to prevent and mitigate their incidence and severity.

[0184] In certain embodiments, the multispecific antibodies or methods used in this disclosure are administered after the subject has been pretreated with standard care therapies such as chemotherapy, immunotherapy, or targeted therapy for progressive metastatic disease.

[0185] In certain embodiments, the multispecific antibodies or methods used in this disclosure are administered to subjects who have not been treated with anti-PD-L1 agents such as anti-PD-L1 antibodies or T-cell agonists.

[0186] In certain embodiments, the cancer is an advanced or metastatic solid tumor. In particular, the cancer may be selected from lung cancer, particularly locally advanced or metastatic NSCLC, or melanoma, particularly cancer that has been previously treated with immune checkpoint therapy and / or is positive for PD-L1. In certain embodiments, the cancer is an advanced or metastatic solid tumor with PD-L1 expression of 1% or more. In certain embodiments, the cancer or subject containing the cancer has either previously received approved anti-PD-1 / PD-L1 immunotherapy and has PD-L1 expression of 1% or more, or the cancer or subject is treatment-naive and has PD-L1 expression of 1% or more.

[0187] In certain embodiments, the cancer is an advanced or metastatic solid tumor, such as locally advanced or metastatic lung cancer and locally advanced or metastatic melanoma. In certain embodiments, the melanoma is such that it

[0188] PD-L1 expression is determined by a clinician skilled in the art, and the entire method is incorporated herein by reference to the exemplary method of de Ruiter et al. (2021). High expression determined by any one of these methods constitutes high expression for the purposes of this disclosure. When assessed by tumor proportion score (TPS), a TPS score of 1% or higher indicates PD-L1 positive cancer. When assessed by combined positive score (CPS), a score of 1% or higher indicates PD-L1 positive cancer. Cancer is considered to have high PD-L1 if it has a PD-L1 expression score of 10% or higher, 20% or higher, 30% or higher, 40% or higher, or 50% or higher, as determined using tumor proportion score (TPS). Cancer is considered to have high PD-L1 if it has a PD-L1 expression score of 5% or higher, 15% or higher, or 20% or higher, as determined using combined positive score (CPS). If evaluated by multiple such methods, the criteria of this disclosure are met if one or more of the methods identify the tumor as PD-L1 positive.

[0189] In certain embodiments, the subjects have not received prior treatment with immune checkpoint inhibitors. In certain embodiments, the subjects have not received prior treatment with immune checkpoint inhibitors, and their cancer is positive for PD-L1 expression. In certain embodiments, the subjects have not received prior treatment with immune checkpoint inhibitors and are being treated for melanoma, particularly locally advanced or metastatic melanoma. In certain embodiments, the subjects have not received prior treatment with immune checkpoint inhibitors and are being treated for lung cancer, particularly non-small cell lung cancer (NSCLC).

[0190] NSCLC can be non-squamous or squamous. In some embodiments, subjects may have previously received treatment based on BRAF, ALK, EGFR, and / or ROS1 status. In certain embodiments, subjects may have ongoing or intolerant treatment. In certain embodiments, NSCLC is unresectable.

[0191] In certain aspects, melanoma is cutaneous melanoma, acral melanoma, or mucosal melanoma. Melanoma can be identified by standard histological or cytological processes. In certain aspects, melanoma is not ocular melanoma or uveal melanoma. In certain aspects, melanoma is stage III or stage IV melanoma (according to standard staging systems such as the American Joint Staging Committee). This may be unresectable and / or may be unresponsive to local therapy.

[0192] In certain manifestations, melanoma may be BRAF-positive. In these manifestations, subjects may have previously received BRAF±MEF targeted therapy, and may have experienced disease progression.

[0193] In certain embodiments, the subject may have previously received or be intolerant to standard therapies for progressive or recurrent / metastatic disease, particularly systemic therapies. Standard therapies may include, for example, chemotherapy, immunotherapy, and targeted regimens. The subject may have received, for example, one, two, three, four, or five regimens, but in particular, four or fewer regimens.

[0194] In certain embodiments, the subject has previously received immune checkpoint therapy, particularly PD-L1 / PD-1 therapy. In some such embodiments, the subject has previously received one or fewer PD-1 therapies for advanced or metastatic cancer. In some such embodiments, the subject may have relapsed after prior therapy, for example, the disease of the subject is advanced or remains stable after at least 6 months of treatment in the best case. In certain embodiments, the subject has not received prior treatment with immune checkpoint inhibitors.

[0195] In some embodiments, the subject may have received monotherapy with a multispecific antibody for 1, 2, 3, 4, 5, or 6 months, particularly 1 or 2 months, before initiating combination therapy.

[0196] In certain aspects, the subject is an adult. In certain aspects, the subject is a mammal, particularly a human.

[0197] In certain embodiments, the subject does not have one or more conditions selected from Burkitt lymphoma, lymphoblastic leukemia / lymphoma, lymphoplasmacytic lymphoma, chronic lymphocytic leukemia, CNS metastasis / lymphoma, carcinomatous meningitis, autoimmune disease, active HBV, active HCV, or HIV. In certain embodiments, the subject has not received prior treatment including 4-1BB agonists, CAR T-cell therapy, or solid organ or allogeneic stem cell transplantation. In certain embodiments, the subject does not have a history of idiopathic pulmonary fibrosis, interstitial lung disease, organizing pneumonia, drug-induced pneumonia, or idiopathic pneumonia or interstitial pneumonia.

[0198] In certain embodiments, the use of the multispecific antibodies or methods of the present disclosure particularly applies to immunodeficient mice carrying breast cancer, more specifically, human MDA-MB-231 tumors, and CD8 + It increases the number of T cells.

[0199] In certain embodiments, the multispecific antibodies or methods used in the present disclosure are determined to be significantly less toxic than the combination of urelumab and atezolizumab, particularly in immunodeficient mice carrying breast cancer, and more specifically, human MDA-MB-231 tumors, in the same studies, etc.

[0200] In certain embodiments, the multispecific antibodies or methods used in this disclosure do not induce graft-versus-host disease.

[0201] As used herein, the term “antibody” means a proteinaceous molecule, such as one belonging to the immunoglobulin class of proteins, that contains one or more domains that bind to an epitope on an antigen, and such domains derive from or share sequence homology with the variable domain of the antibody. Antibodies for therapeutic use are preferably as close as possible to the natural antibody of the target being treated (e.g., a human antibody for a human target). Antibody binding can be expressed in terms of specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by the binding domain. Affinity is a measure of the strength of binding to a particular antigen or epitope. Antibodies such as the multispecific antibodies of this disclosure typically contain a constant domain (Fc portion) of a natural antibody that can be manipulated as described elsewhere herein, for example, to reduce ADCC and / or CDC activity.

[0202] A "multispecific antibody" refers to an antibody that contains at least two binding sites having different antigen or epitope specificity. In certain embodiments, one or more of the antigen-binding sites contain an immunoglobulin VH / VL pair. In certain embodiments, each of the antigen-binding sites contains an immunoglobulin VH / VL pair.

[0203] In certain embodiments, the multispecific antibody according to this disclosure has two or fewer antigen-binding sites. This means that the antigen-binding portion of such multispecific antibody consists of two antigen-binding sites without the presence of additional antigen-binding sites. In certain embodiments, each of the two antigen-binding sites contains an immunoglobulin VH / VL pair.

[0204] In certain embodiments, the VL in each VH / VL pair is similar. In certain embodiments, the VL in each VH / VL pair is identical. In certain embodiments, the multispecific antibody is a full-length antibody having one heavy / light (H / L) chain combination that binds to the extracellular portion of CD137 and one H / L chain combination that binds to the extracellular portion of a member of the B7 family. In certain embodiments, the light chain in the first H / L chain combination is the same as the light chain in the second H / L chain combination. In certain embodiments, the light chains in the first and second H / L chain combinations are identical.

[0205] In certain embodiments, a multispecific antibody is a bispecific antibody.

[0206] The term “bispecific antibody” means that a portion of the antibody binds to one epitope on an antigen, while a second portion binds to either a different epitope on the same antigen or to a different antigen. Different epitopes typically reside on different antigens. However, different epitopes can also reside on the same antigen. Depending on the expression levels, (sub)cellular localization, and stoichiometry of the two antigens recognized by the bispecific antibody, both Fab arms of the antibody may or may not bind to their epitopes simultaneously. One arm of a bispecific antibody typically contains the variable domain of one antibody, and the other arm contains the variable domain of the other antibody (i.e., one arm of a bispecific antibody is formed by one heavy chain paired with one light chain, and the other arm is formed by a different heavy chain paired with a light chain). In certain embodiments, the heavy chain variable regions of the bispecific antibody of this disclosure are different from each other, but the light chain variable regions are the same in the bispecific antibody of this disclosure. Bispecific antibodies in which different heavy chain variable regions are associated with the same or common light chain variable region are also called bispecific antibodies having a common light chain variable region (cLcv). In certain embodiments, the light chain constant region is also the same. Such bispecific antibodies are referred to as having a common light chain (cLc).

[0207] A particular preferred embodiment is an immunoglobulin having an IgG format, which offers the advantage that the half-life of the divalent conjugated molecule / antibody / variant according to this disclosure is typically longer compared to polyvalent compounds. Furthermore, the immunogenicity of the divalent conjugated molecule according to this disclosure is typically lower compared to polyvalent compounds. The molecules / antibodies / variants according to these embodiments preferably maintain the structure of natural IgG and thus preserve all the benefits associated with the structure of natural IgG.

[0208] The “variants” of antibodies or multispecific antibodies described herein include functional moieties, derivatives, and / or analogs of antibodies or multispecific antibodies. A variant may be a fragment of an antibody, e.g., a Fab fragment. A variant may be a single-stranded variable fragment (scFv). A variant maintains the binding specificity of the antibody. Functional moieties, derivatives, and / or analogs maintain the binding specificity of the antibody. Binding specificity is defined by the ability to bind to the extracellular portions of the first and second membrane proteins, as described herein. A variant may have amino acid insertions, deletions, substitutions, or combinations thereof with respect to a given amino acid sequence (e.g., the SEQ ID NO: 1 in this disclosure), and may have up to 15 amino acid insertions, deletions, substitutions, or combinations thereof with respect to the amino acid sequence of a given SEQ ID NO: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, such as 0, 1, 2, 3, 4, or 5, such as 0, 1, 2, 3, or 4, such as 0, 1, 2, or 3, such as 0, 1, or 2, or 0 or 1.

[0209] As used herein, the term “antigen-binding site” means a site on a binding molecule or antibody that specifically binds to an epitope of an antigen. Such an antigen-binding site is preferably derived from or sharing sequence homology with the variable domain of an antibody, particularly its CDR region. In some preferred embodiments, the antigen-binding site is an immunoglobulin variable domain formed by an immunoglobulin VH / VL pair. In other embodiments, the antigen-binding site is derived from an antibody mimetic, such as an affibody molecule, affiline, affimer, afitin, alpha-body, antikalin, avimer, DARPin, finomer, Knitz domain peptide, or monobody, as described herein earlier.

[0210] The term “full length” as used in this disclosure is defined as including an essentially complete antibody that does not contain one or more artificially added portions having a size of more than 20 amino acid residues, such as an additional antigen-binding site or additional activation site, or an additional ligand or additional ligand-binding site. However, full-length antibodies do not necessarily possess all the functions of an intact antibody. To avoid misunderstanding, a full-length antibody contains two heavy chains and two light chains. Each chain contains a constant (C) and a variable (V) region, which can be classified into domains designated as CH1, CH2, CH3, VH for the heavy chain and CL, VL for the light chain. The heavy chain domains are preferably arranged in the order of the native antibody (VHCH1-CH2-CH3; meaning the VH domain is adjacent to the CH1 domain, followed by the CH2 domain, and then the CH3 domain). The light chain domains are also preferably arranged in the order of the native antibody (VL-CL; meaning the VL domain is adjacent to the CL domain). The antibody binds to the antigen via the variable domain contained in the Fab fragment portion. Antibodies can interact with molecules and cells of the immune system via their constant domain, primarily through the Fc moiety.

[0211] In certain embodiments, full-length IgG antibodies are preferred due to their typical desirable half-life and because, for immunogenic reasons, they are desired to remain entirely close to the (human) molecule. In certain embodiments, the multispecific antibodies of the Disclosure are full-length IgG1, full-length IgG2, full-length IgG3, or full-length IgG4 antibodies.

[0212] Full-length antibodies include antibodies in which mutations may exist that either provide desired characteristics or are merely substitutes for those in the original chain. Such mutations are typically not deletions of substantial portions of any region. However, antibodies in which one or more amino acid residues are inserted, deleted, substituted, or have combinations thereof without essentially altering the antigen-binding properties of the resulting antibody are included in the term "full-length antibody." For example, an IgG antibody may have 1 to 20 amino acid residue insertions, substitutions, deletions, or combinations thereof within its constant region.

[0213] In this specification, unless otherwise specified, the CDRs and amino acid positions assigned to the framework within the heavy chain variable region of an antibody or antibody fragment are designated according to Kabat numbering (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)). Amino acids within the constant region are indicated by the EU numbering system.

[0214] In certain embodiments, “identity percentage (%)” for nucleic acid sequences or amino acid sequences as defined herein is defined as the percentage of residues in a candidate sequence that are identical to residues in a selected sequence after the sequences have been aligned for optimal comparison purposes. To optimize the alignment between these two sequences, gaps may be introduced in either of the two sequences being compared. Such alignment may be performed over the entire length of the sequences being compared. Alternatively, the alignment may be performed over shorter lengths, e.g., about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Alignment may also be performed over individual CDR sequences. Sequence identity is the percentage of perfect match between these two sequences across the reported aligned region.

[0215] In certain embodiments, the comparison of sequences and the determination of the percentage of sequence identity between two sequences are achieved using mathematical algorithms. Those skilled in the art will recognize that several different computer programs are available for aligning two sequences and determining the identity between them (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percentage of sequence identity between two amino acid sequences or nucleic acid sequences can be determined using the Needleman-Wunsch algorithm for aligning two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. In certain embodiments, the NEEDLE program from the EMBOSS package is used to determine the identity percentage of amino acids and nucleic acid sequences (version 2.8.0, EMBOSS: The European Molecular Biology Open Software Suite (2000), Rice, P. Longden, J. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ). In certain embodiments, EBLOSUM62 is used as the substitution matrix for protein sequences. In certain embodiments, DNAFULL is used for DNA sequences. The parameters used are a gap-open penalty of 10 and a gap-elongation penalty of 0.5.

[0216] As described above, after alignment by the NEEDLE program, the percentage of sequence identity between the query sequence and the sequence of this disclosure is calculated as follows: The number of corresponding positions in the alignment represents the number of identical amino acids or identical nucleotides in both sequences divided by the total length of the alignment after subtracting the total number of gaps in the alignment.

[0217] In certain embodiments, at the start of treatment, at least one, two or more, or all of the following inclusion factors IF1-IF20 are met for treatment. The start of treatment means the first administration of the multispecific antibody of this disclosure. In certain embodiments, the subject includes or conforms to all of IF1-IF: IF 1. Having a life expectancy of 12 weeks or more. The device must have an ECOG performance status of IF 2.0 or 1. IF3. Having a locally advanced tumor that has relapsed in response to PD-L1 / PD-1 and is positive for PD-L1 expression (≥1%) on tumors and / or tumor-associated immune cells (based on local trials after completion of the most recent previous therapy line). Having a disease measurable according to IF4.RECISTv1.1 or Lugano criteria. IF5. The patient has previously received standard therapy for an advanced or recurrent / metastatic disease corresponding to the tumor type, and is either intolerant to treatment or has refused standard treatment. IF6. Unless otherwise approved by Medical Monitor, the patient has previously received up to four systemic treatment regimens (including chemotherapy, immunotherapy, and targeted therapy regimens) for an advanced or relapsed / metastatic disease. IF7. Provided that anti-PD-1 therapy is acceptable in combination with chemotherapy, targeted therapy, or other immunotherapy, the patient has previously received up to one course of anti-PD-1 therapy containing an immunotherapy regimen, in the advanced / metastatic setting. Tumor-specific criteria IF8. Subjects with histologically or cytologically confirmed cutaneous melanoma, acral melanoma, or mucosal melanoma, or subjects with histologically or cytologically confirmed melanoma. IF9. Unresectable stage III or stage IV melanoma according to the American Joint Committee on Cancer staging system, in which local therapy is ineffective. IF10. If not previously tested, having a V600 activated BRAF mutation status during the screening period, or consent to BRAF testing. If the IF11.BRAF mutation is positive, the patient must be receiving BRAF±MEK targeted therapy and disease progression must be documented. IF12. Relapse in response to PD-L1 / PD-1 therapy (e.g., progression after more than 6 months of treatment, with the best-case scenario being at least disease stabilization) IF13. Relapse in response to PD-L1 / PD-1 therapy (e.g., progression after more than 6 months of treatment, with the best-case scenario being at least disease stabilization) IF14. PD-L1 / PD-1 therapy is naive IF15. Patients whose progression of anti-PD-1 therapy has been documented, as defined by meeting one of the following criteria: IF15a. Primary refractory: The patient has previously received anti-PD-1 therapy (alone or as part of a combination therapy) for at least 12 weeks in a setting of progressive or metastatic disease, and has PD as the best response to treatment. IF15b. Secondary resistance: In a setting of progressive or metastatic disease, the patient previously received anti-PD-1 therapy (alone or as part of a combination) and achieved complete response (CR), partial response (PR), or stable disease (SD), but subsequently developed disease (PD) while receiving anti-PD-1 therapy (PD was confirmed at least 4 weeks [28 days] later). IF16. Subjects with a histologically or cytologically confirmed diagnosis of NSCLC (either non-squamous or squamous epithelial). IF16a. If the tumor is of the non-squamous type only, document the results of testing for EGFR, ALK, BRAF, and ROS1 mutations or gene rearrangements; however, if mutations or gene rearrangements are present, the patient is either progressing to or intolerant of targeted therapy. IF17. Unresectable advanced or metastatic NSCLC according to the AJCC staging system, ineffective with local therapy. IF18. Patients who have relapsed to PD-L1 / PD-1 therapy (e.g., disease progression after more than 6 months of treatment, with the best response being at least stable disease) IF19. Patients who have relapsed to PD-L1 / PD-1 therapy (e.g., progression after more than 6 months of treatment, with the best response being at least stable disease), or who are PD-L1 / PD-1 therapy naive. IF20. Patients whose progression of anti-PD-1 therapy has been documented by meeting one of the following criteria: IF20a. In cases of primary refractory disease, defined as progressive or metastatic, patients who have previously received at least 12 weeks of anti-PD-1 therapy (alone or as part of a combination) and whose best response to treatment is PD. IF20b. In cases of secondary resistance, patients with progressive or metastatic disease who previously received anti-PD-1 therapy (alone or as part of a combination) and achieved complete response (CR), partial response (PR), or stable disease (SD), but subsequently experienced disease progression (PD) while receiving anti-PD-1 therapy (PD was observed at least 4 weeks [28 days] later).

[0218] In certain aspects, the target of treatment is based on any one or more factors selected from the group consisting of IF1 to IF20.

[0219] In certain embodiments, at the start of treatment, at least one, two or more, or all of the following exclusion factors EF1-EF23 are met, making the patient eligible for treatment: EF1. Prior to administration of the first dose of the multispecific antibody of this disclosure, the patient has received treatment with an anticancer agent or investigational drug within the following intervals: EF1a: If the subject does not have radiation pneumonitis as a result of treatment, and a one-week washout period is permitted, then at least 14 days for chemotherapy (6 weeks for mitomycin C and nitrosourea), targeted small molecule therapy, or radiotherapy. EF1b: For previous antibodies used in anticancer therapy, at least 14 or 28 days, EF1c: In other drugs with long half-lives (e.g., more than 5 days), administration of the multispecific antibody of this disclosure is before the 5th half-life requires approval from a medical monitor. EF1d: In the case of radioimmunotherapy, at least 10 weeks, or EF1e: Individuals who have not undergone a similar SCT within the past 6 months, or who have not undergone a self-administered SCT within the past 3 months. EF2: Subjects who are expected to have alopecia and stable neuropathy (grade 2 or less), and who have not recovered to grade 1 or less or to baseline from toxic effects of prior therapies (including prior immunotherapy) and / or complications from prior surgical interventions prior to the first administration of the multispecific antibody of this disclosure. EF3. Subjects with grade 1 or higher hepatotoxicity from previous anti-PD-1 therapy (± anti-CTLA-4 therapy), provided that baseline transaminase and bilirubin levels are outside the normal limit. EF4. Subject to the following conditions, where grade 3 or higher adverse events are acceptable, patients with a prior history of grade 3 or higher immune-mediated adverse events with anti-PD-1 therapy: grade 3 rash resolved with topical therapy; immune-mediated adrenal insufficiency, type 1 diabetes, or other endocrine abnormalities resulting from previous immunotherapy that are medically stable and adequately managed with stable doses of replacement therapy; and asymptomatic amylase or lipase elevation that does not require treatment interruption. EF5. Having a history of any grade of immune-mediated ocular adverse event. EF6. Subjects with the test values ​​defined in the table below during screening. [Table 1] EF7. Subjects with arrhythmias who are taking antiarrhythmic drugs and have sinus rhythm as confirmed by ECG screening, and who have a known history of left ventricular ejection fraction less than 50%, unstable angina within 6 months on day 1 of the cycle, acute myocardial infarction, congestive heart failure of New York Heart Association class III or IV, or arrhythmias requiring therapy. EF7. In the opinion of the principal investigator, subjects with a clinically significant history of or presence of ECG. EF8. Patients with immuno-related toxicity during checkpoint inhibitor therapy prior to the recommendation of permanent discontinuation of treatment (according to the product label or consensus guidelines), or any immuno-related toxicity (excluding endocrine disorders well controlled by replacement hormones) that requires intensive or prolonged immunosuppression to manage. EF9. Subjects with a history of idiopathic pulmonary fibrosis, interstitial lung disease, organizing pneumonia (e.g., bronchiolitis obliterans), drug-induced pneumonia, or idiopathic pneumonia, or evidence of interstitial pneumonia on screening chest CT scans. EF10. Subjects with a history of uncontrolled asthma or chronic obstructive pulmonary disease, provided that a history of radiation pneumonitis (fibrosis) in a radiation field is acceptable. EF11. Subjects with previously treated brain metastases are EF11a, subject to the condition that they have known active CNS metastases / lymphoma and / or carcinomatous meningitis: radiologically stable (i.e., no evidence of progression by repeated imaging for at least 28 days prior to the first dose of the multispecific antibody of this disclosure), EF11b: no need for steroid treatment for at least 14 days prior to the first dose of the multispecific antibody of this disclosure, or EF11c: clinically stable with any neurological signs or symptoms returned to baseline, provided that carcinomatous meningitis is excluded regardless of clinical stability. EF12: Subjects with evidence of cerebral edema, or subjects who have received radiotherapy for the central nervous system (CNS) less than 28 days prior. EF13: Subjects with vitiligo, resolved childhood asthma / atopic dermatitis, hypothyroidism stable on hormone replacement, controlled asthma, type 1 diabetes, Graves' disease, or Hashimoto's disease, or subjects who have received approval from a medical monitor and meet all other eligibility criteria, who have active or inactive autoimmune disease or syndrome (e.g., rheumatoid arthritis, moderate or severe psoriasis, multiple sclerosis, inflammatory bowel disease) requiring systemic treatment in the past two years, or who are receiving systemic therapy for an autoimmune or inflammatory disease (i.e., with the use of disease modifiers, corticosteroids, or immunosuppressants). EF14: Subjects who have used systemic corticosteroids (prednisone or equivalent at a dose of 10 mg / day or more) within 7 days prior to the administration of the first dose of the multispecific antibody of this disclosure, provided that the use of inhaled or topical or systemic corticosteroids for imaging processing is permitted and the use of physiological corticosteroid replacement therapy may be approved after consultation with a medical monitor. EF15. Administration of live vaccines, including but not limited to measles, mumps, rubella, varicella, yellow fever, rabies, BCG, and typhoid vaccines, within 30 days prior to administration of the first dose of the multispecific antibody or pembrolizumab of this disclosure. If the EF16.COVID-19 live vaccine is available, you should consult a medical monitor before administering it. EF17. Assuming that intranasal influenza vaccines, which are attenuated live vaccines, are not expected, injectable seasonal influenza vaccines are generally expected to be inactivated virus vaccines. EF18. Having an active infection requiring systemic therapy. EF19. Having a history of solid organ or allogeneic stem cell transplantation. Subjects who have previously cleared EF20.HBV infection (defined as negative for hepatitis B surface antigen, positive for hepatitis B surface antibody, and positive for hepatitis B core antibody), subjects who have been vaccinated against HBV and have no prior history of HBV infection, and who are antibody-positive for hepatitis B surface antigen based solely on previous exposure, and subjects who have undetectable HCV RNA levels and, if permissible, have undergone and completed treatment for hepatitis C intended to eradicate the virus, and who are hepatitis C antibody-positive, are eligible to participate, provided that they have active HBV or HCV infection requiring treatment, but their hepatitis B virus DNA and HCV RNA are undetectable. EF21. Having a known history of HIV (HIV 1 / 2 antibody). EF22. Having a known hypersensitivity or severe reaction to any component of the multispecific antibody of this disclosure, pembrolizumab, or formulation component. EF23. From the time of the screening visit until 90 days after the last dose of the multispecific antibody of this disclosure, the participant must be pregnant or lactating, or expect to conceive or become a father within the planned period of the study.

[0220] In certain embodiments, the target of treatment is based on any one or more factors selected from the group consisting of EF1 to EF23. In certain embodiments, the target of treatment is based on all of factors EF1 to EF23.

[0221] Clause Certain aspects of this disclosure are defined in the following clauses. 1. A multispecific antibody for use in a method of cancer treatment in a subject requiring cancer treatment, comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, wherein the treatment further comprises administering a PD-L1 or PD-1 inhibitor. 2. A method for treating cancer in a subject requiring cancer treatment, comprising administering a multispecific antibody, which includes an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, and a PD-1 or PD-L1 inhibitor to the subject requiring cancer treatment. 3. A multispecific antibody for use according to claim 1, or the method according to claim 2, wherein the multispecific antibody is administered sequentially or separately at the same time as a PD-1 or PD-L1 inhibitor. 4. A multispecific antibody or method for use according to any one of the prior claims, wherein the cancer is an advanced or metastatic solid tumor, such as selected from locally advanced or metastatic lung cancer and locally advanced or metastatic melanoma. 5. A multispecific antibody or method for use according to any one of the prior claims, wherein the cancer is NSCLC. 6. A multispecific antibody or method for use according to claim 4, wherein the melanoma is selected from cutaneous melanoma, acral melanoma, or mucosal melanoma. 7. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the cancer has relapsed in response to PD-1 / PD-L1 therapy and / or is positive for PD-L1 expression. 8. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the PD-1 or PD-LI inhibitor is an antibody, or a variant or functional fragment thereof. 9. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the PD-1 or PD-LI inhibitor is a PD-1 inhibitor. 10. A multispecific antibody or method for use as described in Clause 9, wherein the PD-1 inhibitor is selected from nivolumab, pembrolizumab, semiprimab, penprimab, retifanlimab, cintilimab, tislerizumab, tripalimab, and dostarimab. 11. A multispecific antibody or method for use as described in Clause 10, wherein the PD-1 inhibitor is pembrolizumab. 12. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the PD-1 or PD-LI inhibitor is a PD-L1 inhibitor. 13. A multispecific antibody or method for use as described in Clause 12, wherein the PD-L1 inhibitor is selected from atezolizumab, avelumab, and durvalumab. 14. A multispecific antibody or method for use as described in any of the preceding clauses, in which the multispecific antibody is administered in doses of 10-600 mg, 15-600 mg, 25-600 mg, 10-300 mg, 15-300 mg, or 25-300 mg, such as 10-150 mg, 10-100 mg, 10-75 mg, 15-150 mg, 15-100 mg, 15-75 mg, 25-150 mg, 25-100 mg, or 25-75 mg, such as 10-50 mg, 15-50 mg, 25-50 mg, or 50-100 mg, in doses of 10-1200 mg, 15-1200 mg, or 25-1200 mg. 15. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the multispecific antibody is administered in doses of approximately 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, or 75 mg. 16. A multispecific antibody or method for use as described in any of the preceding clauses, wherein a PD-1 or PD-L1 inhibitor is administered in doses of approximately 300-500 mg, such as 325-475 mg, 350-450 mg, or 375-425 mg. 17. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the multispecific antibody is administered every 14 days. 18. A multispecific antibody or method for use as described in any of the preceding clauses, wherein a PD-1 or PD-L1 inhibitor is administered every 4, 5, 6, 7, or 8 weeks. 19. A multispecific antibody or method for use as described in Clause 18, wherein a PD-1 or PD-L1 inhibitor is administered every six weeks. 20. A multispecific antibody or method for use described in any of the preceding clauses, in which a multispecific antibody and a PD-1 or PD-L1 inhibitor are administered in the dose combinations shown in the table below: [Table 2-1] [Table 2-2] 21. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the multispecific antibody is administered intravenously. 22. A multispecific antibody or method for use as described in any preceding clause, wherein the multispecific antibody is administered once every two weeks. 23. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the second membrane protein is not a member of the TNF receptor superfamily. 24. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the second membrane protein is a member of the B7 family. 25. A multispecific antibody or method for use as described in any preceding clause, wherein the second membrane protein is PD-L1, PD-L1, or PD-L2. 26. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the multispecific antibody comprises one antigen-binding site that binds to the PD-1 binding domain of PD-L1. 27. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the multispecific antibody comprises one antigen-binding site that binds to the CD137L-binding domain of CD137. 28. A multispecific antibody or method for use as described in any preceding clause, comprising one antigen-binding site that blocks the binding of a ligand to CD137 or binds to an extracellular ligand-blocking site of CD137, such as a CD137L blocking site. 29. A multispecific antibody or method for use as described in any of the preceding clauses, defined as a variable domain, in which a variable domain that binds to the extracellular portion of CD137 is present in a bivalent monospecific antibody format containing two of the variable domains that bind to CD137, and either does not stimulate the activity of CD137 on a cell, or stimulates it at a reduced level compared to one of the variable domains as part of a bispecific antibody having a second variable domain that binds to a tumor-associated antigen such as PD-L1, such as a member of the B7 family. 30. A multispecific antibody or method for use as described in any prior clause, in which, in the presence of a first cell expressing CD137 and a second cell expressing PD-L1, the activity of CD137 on the cell can be stimulated if the variable domain that binds to the extracellular portion of CD137 is combined with the second variable domain that binds to PD-L1 in the multispecific antibody. 31. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the multispecific antibody can bind to CD137 and PD-L1 simultaneously, for example. 32. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the multispecific antibody induces or activates CD137 signaling only in the presence of PD-L1 expressing cells. 33. A multispecific antibody or method for use as described in any preceding clause, wherein the antigen-binding site of the multispecific antibody comprises one immunoglobulin variable domain that binds to CD137 and one immunoglobulin variable domain that binds to the extracellular portion of a second membrane protein. 34. A multispecific antibody or method for use described in any of the preceding clauses, wherein the multispecific antibody is a full-length antibody. 35. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the multispecific antibody is an IgG1 molecule that does not have Fc effector function. 36. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the second membrane protein is not significantly expressed by T cells. 37. A multispecific antibody or method for use as described in any preceding clause, wherein the second membrane protein is present on the cell membrane as part of a multimeric membrane protein comprising two or more of the second membrane proteins. 38. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the second membrane protein is present on the cell membrane as part of a homodimer or homotrimer. 39. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region that binds to the extracellular portion of CD137, which includes an amino acid sequence represented by SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 34, or SEQ ID NO: 52, or a CDR3 region having a variant thereof. 40. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region that binds to the extracellular portion of CD137, which includes a CDR2 region having the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 51, or a variant thereof. 41. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region that binds to the extracellular portion of CD137, which includes an amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 32, or SEQ ID NO: 50, or a CDR1 region having a variant thereof. 42. A multispecific antibody or method for use as described in any preceding clause, wherein the antibody comprises a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 46, or SEQ ID NO: 49, or a variant thereof, which binds to the extracellular portion of CD137. 43. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region including an amino acid sequence represented by SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 102, or SEQ ID NO: 106, which binds to the extracellular portion of PD-L1, or a CDR3 region having a variant thereof. 44. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region including an amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 94, SEQ ID NO: 101, or SEQ ID NO: 105, or a CDR2 region having a variant thereof, which binds to the extracellular portion of PD-L1. 45. A multispecific antibody according to any of the preceding clauses, wherein the antibody comprises a heavy chain variable region including an amino acid sequence represented by SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, or SEQ ID NO: 93, or a CDR1 region having a variant thereof, which binds to the extracellular portion of PD-L1. 46. ​​A multispecific antibody or method for use as described in any preceding clause, wherein the antibody comprises a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 100, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 107, or a variant thereof, which binds to the extracellular portion of PD-L1. 47. A multispecific antibody or method for use as described in any preceding clause, wherein the multispecific antibody comprises CDR1, 2, and 3 of MF6797 and CDR1, 2, and 3 of MF7702 or their variants. 48. A multispecific antibody or method for use as described in any preceding clause, wherein the multispecific antibody comprises SEQ ID NO: 49 and SEQ ID NO: 67, or variants thereof. 49. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the cancer is Merkel cell carcinoma. 50. A multispecific antibody for use in a method of treating cancer in a subject requiring cancer treatment, wherein the antibody is A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 50, CDR2 having the amino acid sequence shown in SEQ ID NO: 51, and CDR3 having the amino acid sequence shown in SEQ ID NO: 52, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 40, CDR2 having the amino acid sequence shown in SEQ ID NO: 41, and CDR3 having the amino acid sequence shown in SEQ ID NO: 42, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 21, CDR2 having the amino acid sequence shown in SEQ ID NO: 22, and CDR3 having the amino acid sequence shown in SEQ ID NO: 23, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 32, CDR2 having the amino acid sequence shown in SEQ ID NO: 33, and CDR3 having the amino acid sequence shown in SEQ ID NO: 34, comprising a binding domain that binds to CD137, and / or Antibodies, A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 68, CDR2 having the amino acid sequence shown in SEQ ID NO: 55, and CDR3 having the amino acid sequence shown in SEQ ID NO: 56, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 93, CDR2 having the amino acid sequence shown in SEQ ID NO: 94, and CDR3 having the amino acid sequence shown in SEQ ID NO: 95, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 93, CDR2 having the amino acid sequence shown in SEQ ID NO: 101, and CDR3 having the amino acid sequence shown in SEQ ID NO: 102, or It includes a variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 90, CDR2 having the amino acid sequence shown in SEQ ID NO: 79, and CDR3 having the amino acid sequence shown in SEQ ID NO: 91, and a binding domain that binds to PD-L1. Each of the individual sequence numbers has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, or substitutions, or combinations thereof. A multispecific antibody intended for simultaneous, sequential, or separate administration with a PD-1 or PD-L1 inhibitor. 51. Multispecific antibodies for use as described in Clause 49, administered in doses of 10-300 mg, 15-300 mg, or 25-300 mg, such as 25-150 mg or 25-100 mg, or 50-100 mg. 52. A multispecific antibody for use as described in Clause 50 or 51, wherein the cancer is Merkel cell carcinoma. 53. A method for treating cancer in a person requiring cancer treatment, comprising a multispecific antibody, A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 50, CDR2 having the amino acid sequence shown in SEQ ID NO: 51, and CDR3 having the amino acid sequence shown in SEQ ID NO: 52, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 40, CDR2 having the amino acid sequence shown in SEQ ID NO: 41, and CDR3 having the amino acid sequence shown in SEQ ID NO: 42, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 21, CDR2 having the amino acid sequence shown in SEQ ID NO: 22, and CDR3 having the amino acid sequence shown in SEQ ID NO: 23, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 32, CDR2 having the amino acid sequence shown in SEQ ID NO: 33, and CDR3 having the amino acid sequence shown in SEQ ID NO: 34, comprising a binding domain that binds to CD137, and / or Antibodies, A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 68, CDR2 having the amino acid sequence shown in SEQ ID NO: 55, and CDR3 having the amino acid sequence shown in SEQ ID NO: 56, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 93, CDR2 having the amino acid sequence shown in SEQ ID NO: 94, and CDR3 having the amino acid sequence shown in SEQ ID NO: 95, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 93, CDR2 having the amino acid sequence shown in SEQ ID NO: 101, and CDR3 having the amino acid sequence shown in SEQ ID NO: 102, or It includes a variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 90, CDR2 having the amino acid sequence shown in SEQ ID NO: 79, and CDR3 having the amino acid sequence shown in SEQ ID NO: 91, and a binding domain that binds to PD-L1. A multispecific antibody in which each individual sequence number has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, or substitutions, or combinations thereof, A method comprising administering a PD-1 or PD-L1 inhibitor to a subject in need of cancer treatment. 54. The method according to Clause 53, wherein the multispecific antibody is administered in doses of 10-300 mg, such as 25-150 mg or 25-100 mg, or 50-100 mg. 55. A multispecific antibody or method for use according to any one of the clauses 50 to 54, wherein the cancer is an advanced or metastatic solid tumor. 56. A multispecific antibody or method for use as described in Clause 55, wherein the advanced or metastatic solid tumor is selected from advanced or metastatic lung cancer, particularly non-small cell lung cancer (NSCLC), and advanced or metastatic melanoma. 57. A multispecific antibody for use in a method of treating cancer in a subject requiring cancer treatment, wherein the antibody is A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 50, CDR2 having the amino acid sequence shown in SEQ ID NO: 51, and CDR3 having the amino acid sequence shown in SEQ ID NO: 52, or A variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 40, CDR2 having the amino acid sequence shown in SEQ ID NO: 41, and CDR3 having the amino acid sequence shown in SEQ ID NO: 42, or A variable domain comprising CDR1 having the amino acid sequence shown by SEQ ID NO: 21, CDR2 having the amino acid sequence shown by SEQ ID NO: 22, and CDR3 having the amino acid sequence shown by SEQ ID NO: 故23, or A variable domain comprising CDR1 having the amino acid sequence shown by SEQ ID NO: 32, CDR2 having the amino acid sequence shown by SEQ ID NO: 33, and CDR3 having the amino acid sequence shown by SEQ ID NO: 34, and a binding domain that binds to CD137, and / or The antibody is A variable domain comprising CDR1 having the amino acid sequence shown by SEQ ID NO: 68, CDR2 having the amino acid sequence shown by SEQ ID NO: 55, and CDR3 having the amino acid sequence shown by SEQ ID NO: 56, or A variable domain comprising CDR1 having the amino acid sequence shown by SEQ ID NO: 93, CDR2 having the amino acid sequence shown by SEQ ID NO: 94, and CDR3 having the amino acid sequence shown by SEQ ID NO: 95, or A variable domain comprising CDR1 having the amino acid sequence shown by SEQ ID NO: 93, CDR2 having the amino acid sequence shown by SEQ ID NO: 101, and CDR3 having the amino acid sequence shown by SEQ ID NO: 102, or A variable domain comprising CDR1 having the amino acid sequence shown by SEQ ID NO: 90, CDR2 having the amino acid sequence shown by SEQ ID NO: 79, and CDR3 having the amino acid sequence shown by SEQ ID NO: 91, and a binding domain that binds to PD-L1, Each of the individual SEQ ID NOs has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, or substitutions, or combinations thereof, The multispecific antibody is for simultaneous, subsequent, or separate administration with a PD-1 or PD-L1 inhibitor, A multispecific antibody wherein the cancer selects a progressive or metastatic solid tumor. 58. A multispecific antibody or method for use according to any one of clauses 50 to 57, wherein the multispecific antibody is administered intravenously. 59. A multispecific antibody or method for use as described in any one of clauses 50 to 58, wherein the multispecific antibody is administered once every two weeks. 60. A multispecific antibody or method for use according to any one of the clauses 50 to 59, wherein the multispecific antibody comprises CDR1, 2, and 3 of MF6797 and CDR1, 2, and 3 of MF7702 or variants thereof. 61. A multispecific antibody or method for use as described in Clause 60, wherein the multispecific antibody comprises SEQ ID NO: 49 and SEQ ID NO: 67, or variants thereof. 62. A multispecific antibody or method for use according to any one of the clauses 50 to 59, wherein the multispecific antibody comprises CDR1, 2, and 3 of MF6783 and CDR1, 2, and 3 of MF5542 or variants thereof. 63. A multispecific antibody or method for use as described in Clause 62, wherein the multispecific antibody comprises SEQ ID NO: 1 and SEQ ID NO: 92, or variants thereof. 64. A multispecific antibody or method for use according to any one of the clauses 50 to 59, wherein the multispecific antibody comprises CDR1, 2, and 3 of MF6754 and CDR1, 2, and 3 of MF5561 or variants thereof. 65. A multispecific antibody or method for use as described in Clause 64, wherein the multispecific antibody comprises SEQ ID NO: 20 and SEQ ID NO: 100, or variants thereof. 66. A multispecific antibody or method for use according to any one of the clauses 50 to 59, wherein the multispecific antibody comprises CDR1, 2, and 3 of MF6785 and CDR1, 2, and 3 of MF5439 or variants thereof. 67. A multispecific antibody or method for use as described in Clause 66, wherein the multispecific antibody comprises SEQ ID NO: 31 and SEQ ID NO: 89, or variants thereof. 68. A multispecific antibody or method for use according to any one of the clauses 50 to 59, wherein the multispecific antibody comprises CDR1, 2, and 3 of MF6795 and CDR1, 2, and 3 of MF5442 or variants thereof. 69. A multispecific antibody or method for use as described in Clause 68, wherein the multispecific antibody comprises SEQ ID NO: 9 and SEQ ID NO: 92, or variants thereof. 70. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the antibody comprises the CDR1, CDR2, and CDR3 sequences of a common light chain variable domain having the amino acid sequence shown in SEQ ID NO: 110, or a common light chain having the amino acid sequence shown in SEQ ID NO: 109, or variants thereof, and the CDR nomenclature or numbering is by IMGT. 71. A multispecific antibody or method for use as described in any preceding clause, wherein the antibody comprises a common light chain variable domain having the amino acid sequence shown in SEQ ID NO: 110, or a common light chain having the amino acid sequence shown in SEQ ID NO: 109, or a variant thereof. 72. A multispecific antibody or method for use as described in any preceding clause, comprising a heavy chain constant domain 1 (CH1) having the amino acid sequence shown in SEQ ID NO: 112, a heavy chain constant domain 2 (CH2) having the amino acid sequence shown in SEQ ID NO: 114, a heavy chain constant domain 3 (CH3) having the amino acid sequence shown in SEQ ID NO: 115, and a heavy chain constant domain 3 (CH3) having the amino acid sequence shown in SEQ ID NO: 116, or variants thereof. 73. A multispecific antibody or method according to any of the clauses 50 to 72, wherein the PD-1 or PD-L1 inhibitor is a PD-1 inhibitor. 74. A multispecific antibody or method according to Clause 73, wherein the PD-1 inhibitor is selected from nivolumab, pembrolizumab, semiprimab, penprimab, retifanlimab, cintilimab, tislerizumab, tripalimab, and dostarimab. 75. The multispecific antibody or method according to Clause 74, wherein the PD-1 inhibitor is pembrolizumab. 76. A multispecific antibody or method according to any of clauses 50 to 72, wherein the PD-1 or PD-L1 inhibitor is a PD-L1 inhibitor. 77. A multispecific antibody or method according to Clause 76, wherein the PD-L1 inhibitor is selected from atezolizumab, avelumab, and durvalumab. 75. A kit of parts comprising a multispecific antibody as defined in any one of the preceding clauses, and instructions for the use of the multispecific antibody in combination with a PD-1 or PD-L1 inhibitor. 79. A kit of the parts described in Clause 78, further comprising a PD-1 or PD-L1 inhibitor. 80. The kit according to clause 78 or 79, wherein the PD-1 or PD-L1 inhibitor is a PD-1 inhibitor. 81. The kit described in Clause 80, wherein the PD-1 inhibitor is selected from nivolumab, pembrolizumab, semiprimab, and dostarimab. 82. The kit described in Clause 81, wherein the PD-1 inhibitor is pembrolizumab. 83. The kit according to Clause 78 or 79, wherein the PD-1 or PD-L1 inhibitor is a PD-L1 inhibitor. 84. The kit described in Clause 83, wherein the PD-L1 inhibitor is selected from atezolizumab, avelumab, and durvalumab. The kit described in any of the preceding clauses 78 to 84 further includes instructions for the use of multispecific antibodies in doses of 85.10-75 mg, or 25-75 mg, or 25-50 mg, or 25-40 mg, or 25-30 mg, or 10 mg, or 25 mg, or 30 mg, or 40 mg, or 50 mg, or 60 mg, or 70 mg, or 75 mg, or in uniform doses. 86. A kit as described in any of Clauses 78-85, which includes instructions for the use of a multispecific antibody in the treatment of any advanced or metastatic solid tumor. 87. A kit of parts as described in Clause 86, wherein the progressive or metastatic solid tumors are selected from locally advanced or metastatic lung cancer, particularly non-small cell lung cancer (NSCLC), and locally advanced or metastatic melanoma. 88. A combination of a multispecific antibody, comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, and a PD-1 or PD-L1 inhibitor, for use in the treatment of cancer in a subject requiring cancer treatment, wherein the multispecific antibody is as defined in either of the preceding clauses, and the inhibitor is as defined in either of the preceding clauses. 89. A PD-1 or PD-L1 inhibitor as defined in any of the preceding clauses for the treatment of cancer, for subjects requiring treatment of cancer, wherein the PD-L1 inhibitor is intended for co-administration or sequential administration with a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein. 90. A multispecific antibody for use in the treatment of cancer in subjects who have been or are scheduled to be administered a PD-1 or PD-L1 inhibitor as defined in any of the preceding clauses, comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein. 91. A PD-1 or PD-L1 inhibitor, as defined in any of the clauses, for use in the treatment of cancer in patients who have received or are scheduled to receive a multispecific antibody, comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein. [Examples]

[0222] Example 1: A multispecific antibody that binds to PD-L1 and CD137. Multispecific antibodies containing the heavy chain variable region described in Table 1 were obtained as described in WO2018 / 056821. [Table 3] [Table 4]

[0223] Example 2 - Phase 1, open-label, dose-escalation, safety, tolerability, and preliminary efficacy study of MCLA-145 in combination with pembrolizumab in participants with advanced or metastatic malignant tumors. Treatment group and duration: A non-blinded, non-randomized, Phase 1 study was initiated to determine the safety, tolerability, and preliminary efficacy of a bispecific antibody targeting CD137 and PD-L1, in combination with a PD-1 or PD-L1 inhibitor, in adult participants with progressive or metastatic malignancies.

[0224] The following bispecific antibodies are suitable for use in this study and in the methods of the present disclosure: MF6797×MF7702 such as MF6797×MF7702, MF6763×MF5442, MF6754×MF5561, MF6785×MF5439, and MF6785×MF5442. Each bispecific antibody comprises two VHs designated by MF numbers capable of binding to CD137 and PD-L1 respectively, an Fc region having a KK / DE CH3 heterodimerization domain shown by SEQ ID NO: 115 and SEQ ID NO: 116 respectively, a CH2 domain shown by SEQ ID NO: 114, a CH1 domain shown by SEQ ID NO: 112, and a common light chain shown by SEQ ID NO: 109. In particular, the bispecific antibody comprising the heavy chain sequence of MF6797×MF7702 is suitable for use in the studies and methods of the present disclosure.

[0225] A dose escalation study was conducted to determine the MTD and / or RDE of a bispecific antibody in combination with a fixed dose of a PD-1 inhibitor in participants with progressive or recurrent / metastatic solid tumors. Participants were administered escalating doses of one of the above exemplary bispecific antibodies, further referred to herein as the "study antibody", every two weeks until the MTD or RDE was reached. The duration of each treatment cycle while escalating the dose was 28 days. Participants also received a fixed dose of 400 mg of pembrolizumab every six weeks.

[0226] Overall study design This is an open-label, non-randomized, phase 1 study to determine the safety, tolerability, and preliminary efficacy of MCLA-145 in adult participants with advanced or metastatic malignancies, and will be conducted in two parts: dose escalation and dose expansion. MCLA-145, in combination with pembrolizumab administered at a uniform dose every 6 weeks, will be administered intravenously at a uniform dose over 2 hours every 14 days in a 28-day cycle.

[0227] Part 1: Dosage Graduation Part 1 is a dose escalation to determine the maximum tolerated dose (MTD) and / or recommended expanded dose (RDE) of MCLA-145 administered every 14 days in combination with pembrolizumab to participants with advanced or metastatic solid tumors.

[0228] Participants will be treated with MCLA-145 at a dose level of 10 mg in combination with a fixed dose of pembrolizumab. At each dose level, if no dose-limiting toxicity (DLT) is encountered during the first 28-day cycle, including 3 to 6 evaluable patients, the dose of MCLA-145 will be escalated to the next level. Up to 6 evaluable patients may be included at each dose level until a MTD or RDE is reached, or until determined by the sponsor based on the overall safety of the product.

[0229] Participants will first follow the following schema. [Table 5]

[0230] Further combination doses, including 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 100 mg, 125 mg, and 150 mg, may be explored. Pembrolizumab may also be supplied at 200 mg every three weeks.

[0231] Part 2: Dosage Confirmation / Safety Expansion Part 2 is dose expansion to confirm the dose of MCLA-145 in combination with pembrolizumab through further evaluation of safety, tolerability, pharmacokinetics, preliminary antitumor activity, and functional target engagement. Participants with advanced or metastatic tumors will be enrolled as follows:

[0232] Adult men or women with advanced or metastatic solid tumors that have relapsed against PD1 / PD-L1. Participants with NSCLC and melanoma that have relapsed against PD1 / PD-L1 and have a PD-L1 > 1% profile, who are considered by the in-situ investigator to be beneficial to the combination with pembrolizumab.

[0233] Any other tumor histology in Part 1 for which preliminary efficacy has been observed with MCLA-145, or for which new data suggest a response, may be treated in Part 2 after consent from the sponsor's medical monitor. Initial enrollment in the expanded cohort will be limited to 10 or 20 participants per symptom.

[0234] Where supported by new data, enrollment may be limited to PD-L1-positive tumors defined as having a tumor expression of 1% or more, as determined in-situ by any commercially available assay.

[0235] Selection Criteria Participants are eligible to participate in this study only if all of the following criteria apply. Participants with melanoma or NSCLC must meet all criteria listed in all participant sections and applicable tumor-specific criteria sections below to confirm their eligibility.

[0236] All participants The ability and willingness to sign a written ICF for research purposes. You must be at least 18 years old when you sign the informed consent form. Willingness and ability to meet and comply with all protocol requirements, including all scheduled visits and protocol procedures. Average life expectancy of 12 weeks or more. ECOG performance status of 0 or 1. Part 1: Locally advanced or metastatic NSCLC or melanoma that has relapsed in response to PD-L1 / PD-1, and PD-L1 expression is positive (≥1%) on tumor and / or tumor-associated immune cells (based on local trials after completion of the most recent previous therapy line). Part 2: Locally advanced or metastatic NSCLC or melanoma that has relapsed against PD-L1 / PD-1 or is immunotherapy naive, and PD-L1 expression is positive (≥1%) on tumor and / or tumor-associated immune cells (based on local examination after completion of the most recent previous line of therapy). Diseases measurable according to RECIST v1.1 or Lugano criteria. Note: Tumor lesions located in previously irradiated areas or areas that have received other local therapies are considered measurable only if clear progression is demonstrated in the lesion. The patient has previously received standard therapy for an advanced or recurrent / metastatic disease corresponding to the tumor type, and is either intolerant to treatment or has refused standard treatment. Unless otherwise approved by medical monitoring, you have previously received up to four systemic treatment regimens (including chemotherapy, immunotherapy, and targeted therapy regimens) for progressive or recurrent / metastatic disease. Under the criteria of progressive / metastatic disease, patients have previously received up to one course of anti-PD-1 therapy containing an immunotherapy regimen. Anti-PD-1 therapy in combination with chemotherapy, targeted therapy, or other immunotherapy is acceptable. Willingness to undergo tumor biopsies before and during treatment to obtain tumor tissue. Note: In participants from whom tumor tissue cannot be safely obtained, participants may still be enrolled in the study, based on discussions with the medical monitor. Part 2 only: Participants with known PD-L1 status, or participants undergoing a new tumor biopsy to locally assess PD-L1 expression during screening. The willingness to avoid pregnancy or becoming a father, based on the following criteria. Men must agree to take appropriate precautions to avoid becoming fathers (with at least 99% certainty) for 90 days from screening to the last dose of MCLA-145, and must refrain from sperm donation during this period. Approved methods that are at least 99% effective in preventing pregnancy must be communicated to participants, and their understanding must be confirmed. Women of potential pregnancy must have negative serum pregnancy tests at the time of screening and before the first dose on day 1, and must agree to take appropriate precautions to avoid pregnancy (with at least 99% certainty) from screening to 90 days after the last dose of MCLA-145. Approved methods that are at least 99% effective in preventing pregnancy must be communicated to participants, and their understanding must be confirmed. Women of potential pregnancy should refrain from donating oocytes from 30 days before the first dose of MCLA-145 until 90 days after the last dose of MCLA-145. Women who are unable to become pregnant (i.e., surgically infertile due to hysterectomy and / or bilateral oophorectomy, or amenorrhea of ​​12 months or more, and at least 50 years of age) are eligible for registration.

[0237] About combination therapy Patients who have received MCLA145 monotherapy for at least two months are eligible to participate in the study. These patients must not report grade 2 or lower hepatotoxicity during MCLA145 monotherapy.

[0238] Tumor-specific criteria Participants with histologically or cytologically confirmed melanoma: Histologically or cytologically confirmed cutaneous melanoma, acral melanoma, or mucosal melanoma. Note: Participants with ocular melanoma or uveal melanoma will be excluded. Unresectable stage III or stage IV melanoma according to the current AJCC staging system, for which local therapy is ineffective. If not previously tested, documentation of V600 activating BRAF mutation status during the screening period or consent to BRAF testing. If the patient is BRAF mutation-positive, they must be receiving BRAF±MEK targeted therapy and their disease progression must be documented. Part 1 participants in combination therapy: Relapse to PD-L1 / PD-1 therapy (e.g., progression after more than 6 months of treatment, with the best response being at least disease stabilization). Participants in Part 2 of the combination therapy program: Relapse in PD-L1 / PD-1 therapy-naive patients (e.g., progression after more than 6 months of treatment, with the best-case scenario being at least disease stabilization) Part 2 participants receiving monotherapy only: Progression of anti-PD-1 therapy must be documented, defined by meeting one of the following criteria: Primary refractory: Patients must have previously received anti-PD-1 therapy (either alone or as part of a combination with anti-CTLA-4 therapy) for at least 12 weeks in a progressive or metastatic setting, and must have PD as the best response to treatment. Participants who have previously received anti-PD-1 therapy for at least 12 weeks in an adjuvant setting, and those with disease progression, are also expected. Secondary resistance: In the context of progressive or metastatic disease, patients had previously received anti-PD-1 therapy (alone or as part of a combination therapy) and achieved complete response (CR), partial response (PR), or stable disease (SD), but subsequently needed to confirm disease progression (PD) while receiving anti-PD-1 therapy (PD was confirmed at least 4 weeks [28 days] later).

[0239] Participants with histologically or cytologically confirmed NSCLC: The patient has been definitively diagnosed with NSCLC (either non-squamous or squamous epithelial cell carcinoma) based on histological or cytological findings. If the tumor is of the non-squamous type only, documentation of test results for EGFR, ALK, BRAF, and ROS1 mutations or genetic rearrangements is required (molecular testing is not currently part of the diagnostic guidelines for squamous type predominance). If mutations or genetic rearrangements are present, participants must be undergoing targeted therapy or have intolerance to it. Advanced or metastatic NSCLC that is unresectable according to the current AJCC staging system and is ineffective with local therapy. Part 1 participants in combination therapy: Relapse to PD-L1 / PD-1 therapy (e.g., progression after more than 6 months of treatment, with the best response being at least disease stabilization). Participants in Part 2 of the combination therapy program: Relapse in response to PD-L1 / PD-1 therapy (e.g., progression after more than 6 months of treatment, with the best-case scenario being at least disease stabilization) Naive about PD-L1 / PD-1 therapy Part 2 participants only: Progression of anti-PD-1 therapy, defined by meeting one of the following criteria, must be documented: Primary refractory: This category includes progressive or metastatic disease, with a history of at least 12 weeks of prior anti-PD-1 therapy (either alone or as part of a combination therapy), and the patient must have progressive disease (PD) as the best response to treatment. Secondary resistance: In the context of progressive or metastatic disease, patients had previously received anti-PD-1 therapy (alone or as part of a combination therapy) and achieved complete response (CR), partial response (PR), or stable disease (SD), but subsequently needed to confirm disease progression (PD) while receiving anti-PD-1 therapy (PD was confirmed at least 4 weeks [28 days] later).

[0240] Participants with signs of solid tumors confirmed to be MSI-H or dMMR: MSI-H or dMMR solid tumors, determined by a local laboratory using IHC or polymerase chain reaction, and tissue available for confirmation of diagnosis by a central agency are required. Only Part 2 participants considered to be primary refractory to anti-PD-1 therapy are defined as follows: having previously received anti-PD-1 therapy (alone or as part of a combination) for at least 12 weeks in an advanced or metastatic setting, with PD documented as the best response to treatment.

[0241] Exclusion criteria Participants will be excluded from the study if any of the following criteria apply: The following B-cell neoplasms: Burkitt lymphoma, lymphoblastic leukemia / lymphoma, lymphoplasmacytic lymphoma, chronic lymphocytic leukemia. The previous therapy contained a 4-1BB agonist, or the previous therapy was CART cell therapy. Treatment with anticancer drugs or investigational drugs within the following intervals prior to the first dose of MCLA-145: Chemotherapy (6 weeks in the case of mitomycin C and nitrosourea), targeted small molecule therapy, or radiotherapy for at least 14 days. Note: Participants must not have radiation pneumonitis as a result of treatment. A one-week washout period is permitted, with the approval of the sponsor, for palliative radiation for non-CNS diseases. For previous mAbs used in cancer therapy, at least 28 days. Note: Participants who have received bisphosphonates and / or denosumab are eligible for enrollment. For other drugs with long half-lives (e.g., more than 5 days), registration before the fifth half-life requires approval from a medical monitor. In the case of radioimmunotherapy, at least 10 weeks. You must not have undergone a similar SCT within the past six months, or a self-administered SCT within the past three months. Prior to initiating MCLA-145, patients have not recovered to Grade 1 or lower or to baseline from toxic effects of previous therapies (including previous immunotherapy) and / or complications from previous surgical interventions. Note: Alopecia and stable neuropathy (grade 2 or lower) are expected. Previous anti-PD-1 therapy (± anti-CTLA-4 therapy)-related hepatotoxicity is not grade 1 or higher. Only patients receiving combination therapy: Baseline transaminase and bilirubin levels were outside the normal range. A grade 3 or higher immune-mediated adverse event previously experienced with anti-PD-1 therapy. Note: The following Grade 3 or higher adverse events are acceptable: Grade 3 rash resolved with topical therapy; immune-mediated adrenal insufficiency, type 1 diabetes, or other endocrine abnormalities resulting from previous immunotherapy that are medically stable and adequately managed with stable doses of replacement therapy; and asymptomatic amylase or lipase elevation that does not require treatment interruption. A history of any grade of immune-mediated ocular adverse event. Participants with the screening test values ​​defined in the table below. [Table 6-1] [Table 6-2] Clinically significant cardiac disease, including a known history of left ventricular ejection fraction less than 50%, unstable angina within 6 months on day 1 of the cycle, acute myocardial infarction, congestive heart failure of New York Heart Association class III or IV, or arrhythmias requiring therapy. Note: Participants with arrhythmias may be enrolled if they are taking antiarrhythmic medication and their screening ECG shows sinus rhythm. In the opinion of the principal investigator, a clinically significant history or presence of an ECG is required. Immune-related toxicity during checkpoint inhibitor therapy prior to the recommendation of permanent discontinuation of treatment (according to product labeling or consensus guidelines), or any immune-related toxicity requiring intensive or prolonged immunosuppression to manage (excluding endocrine disorders adequately controlled by replacement hormones). A history of idiopathic pulmonary fibrosis, interstitial lung disease, organizing pneumonia (e.g., bronchiolitis obliterans), drug-induced pneumonia, or idiopathic pneumonia, or evidence of interstitial pneumonia on a screening chest CT scan. Participants with a history of uncontrolled asthma or chronic obstructive pulmonary disease will be excluded. Note: A history of radiation pneumonitis (fibrosis) in a radiation field is acceptable. Known active CNS metastases / lymphomas and / or carcinomatous meningitis. Note: Participants with previously treated brain metastases may participate on the condition that 1) they are radiologically stable (i.e., no evidence of progression for at least 28 days by repeated imaging prior to the first dose of MCLA-145 [repeated imaging can be performed during the screening period with the approval of the medical monitor]), 2) they do not require steroid treatment for at least 14 days prior to the first dose of MCLA-145, and 3) they are clinically stable with any neurological signs or symptoms having returned to baseline. This exclusion does not include carcinomatous meningitis, which is excluded regardless of clinical stability. Note: Participants with evidence of cerebral edema or those who have received radiotherapy for the CNS less than 28 days prior will be excluded from the study. Participants who have had active or inactive autoimmune disease or syndrome requiring systemic treatment in the past two years (e.g., rheumatoid arthritis, moderate or severe psoriasis, multiple sclerosis, inflammatory bowel disease), or participants who are receiving systemic therapy for an autoimmune or inflammatory disease (i.e., using disease modifiers, corticosteroids, or immunosuppressants). Note: Participants with vitiligo, resolved childhood asthma / atopic dermatitis, stable hypothyroidism with hormone replacement therapy, controlled asthma, type 1 diabetes, Graves' disease, or Hashimoto's disease, or those approved by a medical monitor, are eligible to participate if they meet all other eligibility criteria. Systemic corticosteroid use (prednisone or equivalent at a dose of 10 mg / day or more) within 7 days prior to the first administration of MCLA-145. Note: Inhalation for imaging procedures, or the use of topical or systemic corticosteroids, is permitted. Note: The use of physiological corticosteroid replacement therapy may be approved after consultation with a medical monitor. Administration of a live vaccine within 30 days of the first dose of MCLA-145 or pembrolizumab. Note: Examples of live vaccines, but not limited to, include: measles, mumps, rubella, varicella, yellow fever, rabies, BCG, and typhoid vaccines. If a live COVID-19 vaccine is available, you should consult your medical monitor before administration. Injectable seasonal influenza vaccines are generally inactivated virus vaccines and are expected, however intranasal influenza vaccines are attenuated live vaccines and are not expected. Active infection requiring systemic therapy. A history of solid organ or allogeneic stem cell transplantation. Active HBV or HCV infection requiring treatment. Hepatitis B virus DNA and HCV RNA must be undetectable. Participants who have cleared previous HBV infection (defined as negative for hepatitis B surface antigen, positive for hepatitis B surface antibody, and positive for hepatitis B core antibody) are eligible to participate in the study. Note: In participants who have previously cleared HBV infection, HBV prophylaxis should be considered at the discretion of the principal investigator. HBV reactivation will be monitored every three cycles by performing HBV viral load and hepatitis B surface antigen serological tests. Additional virological serological tests may be performed at the discretion of the principal investigator. Note: Participants who have been vaccinated against HBV and have positive antibodies to hepatitis B surface antigen based solely on previous exposure, and who have no prior history of HBV infection, are eligible to participate in the study. Note: Participants who have received and completed treatment for hepatitis C intended to eradicate the virus, and who are positive for hepatitis C antibodies, may participate if their HCV RNA levels are undetectable. Known history of HIV (HIV 1 / 2 antibody). HIV testing is not required unless mandated by local health authorities or regulations. Known hypersensitivity or severe reaction to either MCLA-145 or pembrolizumab component, or to any component of the formulation. From the time of your screening visit until 90 days after your last dose of MCLA-145, were you pregnant or breastfeeding, or were you expecting to become pregnant or father within the planned duration of the study? At the discretion of the principal investigator, any condition that interferes with full participation in the study, presents a significant risk to the participant, or interferes with the interpretation of study data, including the administration of MCLA-145 or pembrolizumab.

[0242] Example 3 An 80-year-old male patient was diagnosed with Merkel cell carcinoma, with a PD-L1 score of 1-10%. The patient had received prior treatment with radiotherapy, surgery, and systemic therapy. Treatment with avelumab resulted in progressive disease. Treatment with carboplatin and etoposide resulted in a partial response.

[0243] Subsequently, the patient received a 25 mg dose (25 mg, q3w) every three weeks of a multispecific antibody containing two VHs, designated by MF numbers MF6797 and MF7702, each having an Fc region with a KK / DE CH3 heterodimerization domain (as indicated by SEQ ID NOs. 115 and 116), a CH2 domain (as indicated by SEQ ID NO. 114), a CH1 domain (as indicated by SEQ ID NO. 112), and a common light chain (as indicated by SEQ ID NO. 109), along with a 200 mg dose (200 mg, q3w) of pembrolizumab every three weeks. The disease site was a skin nodule on the foot. Tumor evaluation included the total diameter of the target lesion. After the initial dose, the patient experienced a clinically significant reduction in lesion diameter.

[0244] Example 4 A 40 mg dose of a bispecific antibody, designated by MF numbers MF6797 and MF7702, each containing an Fc region with a KK / DE CH3 heterodimerization domain (as indicated by SEQ ID NOs 115 and 116), a CH2 domain (as indicated by SEQ ID NOs 114), a CH1 domain (as indicated by SEQ ID NOs 112), and a common light chain (as indicated by SEQ ID NOs 109), is administered to subjects with cancer every three weeks (40 mg, q3w) in conjunction with 200 mg (200 mg, q3w) of pembrolizumab, according to Example 2. Tumor evaluation, including the total target lesion diameter, is then performed. After the initial dose, patients may experience a clinically significant reduction in lesion diameter.

[0245] array Sequence ID 1: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTNFAMNWVRRAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVNTAYLQISSLKAEDTAVYYCARDWGVIGGHYMDVWGKGTTVTVSS Sequence ID 2: HCDR1 by Kabat from Sequence ID 1 NFAMN Sequence ID 3: HCDR2 via Kabat from Sequence ID 1 WINTNTGNPTYAQGFTG Sequence ID 4: HCDR3 by Kabat from Sequence ID 1 DWGVIGGHYMDV Sequence ID 5: Multi-strand variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSDGYGPKAFDYWGQGTLVTVSS Sequence ID 6: HCDR1 by Kabat SYGIS Sequence ID 7: HCDR2 by Kabat WISAYNGNTNYAQKLQG Sequence ID 8: HCDR3 by Kabat DSDGYGPKAFDY Sequence ID 9: Double-chain variable region EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPDDSDTRYSPSFQGQVTISADKSSSTAYLQWSSLKASDTAMYYCASFYTGIVGATGAFDVWGQGTTVTVSS Sequence ID 10: HCDR1 by Kabat SYWIG Sequence ID 11: HCDR2 by Kabat IIYPDDSDTRYSPSFQG Sequence ID 12: HCDR3 by Kabat FYTGIVGATGAFDV Sequence ID 13: Heavy Chain Variable Region QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSDAISWVRQAPGQGLEWMGGMIPILGTANYAQKFQGRVTITADRSSTSTAYMELSSLRSEDTAVYYCVRGATYYYGSGTYYSINWFDPWGQGTLVTVSS Sequence ID 14: HCDR1 by Kabat SDAIS Sequence ID 15: HCDR2 by Kabat GMIPILGTANYAQKFQG Sequence ID 16: HCDR3 by Kabat GATYYYGSGTYYSINWFDP Sequence ID 17: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCRASGYTFTNFAMTWVRQAPGQGPEYMGWINTNTGNPTYAQGFTGRFVFSLDTSVNTAYLQISSLKAEDTAVYYCARDWASVMVRGDLDYWGQGTLVTVSS Sequence ID 18: HCDR1 by Kabat NFAMT Sequence ID 19: HCDR3 by Kabat DWASVMVRGDLDY Sequence ID 20: Heavy-chain variable region QVQLVQSGAEVKKPGASVKVSCKVSGYTLSELSIHWVRQAPGKGVEWMGGFYPEDVEPIYARKFQGRVTMTEDTSTDTAYMELNSLRSEDTAVYYCAAEGFDNYGSGIRGNWFDPWGQGTLVTVSS Sequence ID 21: HCDR1 by Kabat ELSIH Sequence ID 22: HCDR2 by Kabat GFYPEDVEPIYARKFQG Sequence ID 23: HCDR3 by Kabat EGFDNYGSGIRGNWFDP Sequence ID 24: Heavy Chain Variable Region EVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQSPGKGLEWMGSFYPEDGETIYAQKFQGRITMTEDTSADTAYMELSSLRSEDTAVYYCATEGVGVIRGNWFDPWGQGTLVTVSS Sequence ID 25: HCDR1 by Kabat ELSMH Sequence ID 26: HCDR2 by Kabat SFYPEDGETIYAQKFQG Sequence ID 27: HCDR3 by Kabat EGVGVIRGNWFDP Sequence ID 28: Heavy Chain Variable Region EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIFPDDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKPSDTAMYYCVRLGGYSGYAEDFVDFWGQGTLVTVSS Sequence ID 29: HCDR2 by Kabat IIFPDDSDTRYSPSFQG Sequence ID 30: HCDR3 by Kabat LGGYSGYAEDFVDF Sequence ID 31 Heavy Chain Variable Region EVQLVQSGAEVKKPGASVKVSCKVSGYTLTKLSMHWVRQAPGKGLEWMGGFEPEDGETINAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATDLRLGASYYYSYMDVWGRGTMVTVSS Sequence ID 32: HCDR1 by Kabat KLSMH Sequence ID 33: HCDR2 by Kabat GFEPEDGETINAQKFQG Sequence ID 34: HCDR3 by Kabat DLRLGASYYYSYMDV Sequence ID 35 Heavy Chain Variable Region QITLKESGPTLVKPTQTLTLSCTFSGFSLSTSGMSVGWIRQPPGKALEWLALIYWNDDKYFSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTATYYCAHTLWGSDDVFDVWGQGTMVTVSS Sequence ID 36: HCDR1 by Kabat TSGMSVG Sequence ID 37: HCDR2 by Kabat LIYWNDDKYFSPSLKS Sequence ID 38: HCDR3 by Kabat TLWGSDDVFDV Sequence ID 39 Heavy Chain Variable Region EVQLVQSGAEVKKPGESLKISCKVSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWHTLKASDTAMYYCARHQGYSFSGSHIDDYWGQGTLVTVSS Sequence ID 40: HCDR1 by Kabat NYWIG Sequence ID 41: HCDR2 by Kabat IIYPGDSDTRYSPSFQG Sequence ID 42: HCDR3 by Kabat HQGYSFSGSHIDDY Sequence ID 43 Heavy Chain Variable Region EVQLVQSGAEVRKPGESLKISCKGSGYSFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTVYLQWSSLKASDTAMYYCARHAGFIITSQNIDDYWGQGTLVTVSS Sequence ID 44: HCDR1 by Kabat TYWIG Sequence ID 41: HCDR2 by Kabat IIYPGDSDTRYSPSFQG Sequence ID 45: HCDR3 by Kabat HAGFIITSQNIDDY Sequence ID 46 Heavy Chain Variable Region EVQLVQSGSELKKPGASVKVSCKASGYTFTNFAMNWVRQAPGQGLEWMGWINTNTGNPTYAQDFTGRFVFSLDTSGNTAYLQISSLKAEDTAVYYCARDWGLVAIGYFDYWGQGTLVTVSS Sequence ID 47: HCDR2 by Kabat WINTNTGNPTYAQDFTG Sequence ID 48: HCDR3 by Kabat DWGLVAIGYFDY Sequence ID 49 Heavy Chain Variable Region QITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVNWIRQPPGEALEWLALIYWNDDTYYSPSLKSRLTITKDTSKNQVVLTMTNMPDPVDTATYYCAHEGIIGFLGGNWFDPWGQGTLVTVSS Sequence ID 50: HCDR1 by Kabat TTGVGVN Sequence ID 51: HCDR2 by Kabat LIYWNDDTYYSPSLKS Sequence ID 52: HCDR3 by Kabat EGIIGFLGGNWFDP Sequence ID 53 Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTSHAMNWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGQGTLVTVSS Sequence ID 54: HCDR1 by Kabat SHAMN Sequence ID 55: HCDR2 by Kabat WINPNTGNPTYAQGFTG Sequence ID 56: HCDR3 by Kabat DRKYVTNWVFAEDFQH Sequence ID 57: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTSHAMNWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAIDRGYMSNWVFAEYFPHWGQGTLVTVSS Sequence ID 58: HCDR3 by Kabat DRGYMSNWVFAEYFPH Sequence ID 59: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCATDRGYISSWVFAEDFQHWGQGTLVTVSS Sequence ID 60: HCDR1 by Kabat SYAMN Sequence ID 61: HCDR3 by Kabat DRGYISSWVFAEDFQH Sequence ID 62: Heavy-chain variable region QVQLVQSGSELKKPGASVKVSCTASGYTFTSYAMNWVRQAPGQRLEWMACVNPNTGSPTYAQGSTGRFVVSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGHGTLVTVSS Sequence ID 63: HCDR2 by Kabat CVNPNTGSPTYAQGSTG Sequence ID 64 Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAMNWVRQAPGQGLEWMGWMNPNTGNPTYAQGSTGRFVVSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGRGTLVTVSS Sequence ID 65: HCDR1 by Kabat NYAMN Sequence ID 66: HCDR2 by Kabat WMNPNTGNPTYAQGSTG Sequence ID 67 Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAINWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGRGTLVTVSS Sequence ID 68: HCDR1 by Kabat NYAIN Sequence ID 69 Heavy Chain Variable Region EVQLVQSGAEVKKPGSSVKVSCKASGDTFNTYSITWVRQAPGQGLEWMGSIVPIFGTINNAQKFQGRVTITADKSANTAYMELSSLRSEDTAVYYCARDNTMVRGVDYYYMDVWGKGTMVTVSS Sequence ID 70: HCDR1 by Kabat TYSIT Sequence ID 71: HCDR2 by Kabat SIVPIFGTINNAQKFQG Sequence ID 72: HCDR3 by Kabat DNTMVRGVDYYYMDV Sequence ID 73 Heavy Chain Variable Region EVQLVQSGAEVKKPGSSVKVSCKASGGIFSTYAISWVRQAPGQGLEWMGGIIPIFDTPNYAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCAKNVRGYSAYDLDYWGQGTLVTVSS Sequence ID 74: HCDR1 by Kabat TYAIS Sequence ID 75: HCDR2 by Kabat GIIPIFDTPNYAQKFQG Sequence ID 76: HCDR3 by Kabat NVRGYSAYDLDY Sequence ID 77 Heavy Chain Variable Region EVQLVQSGAEVKNPGSSVKVSCKATGGTFNTYGTNWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTTTAYMEVSSLRSEDTAVYYCARGGADMGTLDYWGQGTLVTVSS Sequence ID 78: HCDR1 by Kabat TYGTN Sequence ID 79: HCDR2 by Kabat GIIPIFGTANYAQKFQG Sequence ID 80: HCDR3 by Kabat GGADMGTLDY Sequence ID 81 Heavy Chain Variable Region EVQLVQSGAEVMRPGSSVKVSCKASGGIFTYTIIWVRQAPGQGLEWMGGIIPIFDTPNFAQKFQGRLTITADKSTNTAYMELTSLRSEDTAVYYCAREGCNHGVCYPYWGQGTLVTVSS Sequence ID 82: HCDR1 by Kabat TYTII Sequence ID 83: HCDR2 by Kabat GIIPIFDTPNFAQKFQG Sequence ID 84: HCDR3 by Kabat EGCNHGVCYPY Sequence ID 85 Heavy Chain Variable Region QVQLVQSGAEVKKPGSSVKVSCKASGDTFRSYGITWVRQAPGQGLEWMGGIIPIFGTTNYAQKFQGRVTITADKSTSTVYMELSSLRSEDTAVYYCARRRGYSNPHWLDPWGQGTLVTVSS Sequence ID 86: HCDR1 by Kabat SYGIT Sequence ID 87: HCDR2 by Kabat GIIPIFGTTNYAQKFQG Sequence ID 88: HCDR3 by Kabat RRGYSNPHWLDP Sequence ID 89: Heavy Chain Variable Region QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGILWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADISTSTAYMELSSLRSEDTAVYYCARGGGNYYEFVYWGQGTLVTVSS Sequence ID 90 HCDR1 by Kabat TYGIL Sequence ID 91: HCDR3 by Kabat GGGNYYEFVY Sequence ID 92: Double-chain variable region EVQLVQSGAEVKKPGSSVRVSCKASGGTFNTYAINWVRQAPGQGLEWVGRIIPIFDTANYAQKFQGRVTISADKSTTTAYMELSSLRSEDTAVFYCAKDETGYSSSNFQHWGQGTLVTVSS Sequence ID 93 HCDR1 by Kabat TYAIN Sequence ID 94 HCDR2 by Kabat RIIPIFDTANYAQKFQG Kabat sequence number 95 HCDR3 DETGYSSSNFQH Sequence ID 96 Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAINWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGQGTLVTVSS Sequence ID 97: Heavy Chain Variable Region QVQLVQSGAEVKRPGSSVKVSCKASGGTFNTYSITWVRQAPGQGLEWMGIIPVFGTSKYAQKFQDRVTITADKSTNTAYMELSSLRSEDTAVYYCARDPSFSSSSGWFDPWGQGTLVTVSS Sequence ID 98 HCDR2 by Kabat GIIPVFGTSKYAQKFQD Sequence ID 99: HCDR3 by Kabat DPSFSSSSGWFDP Sequence ID 100 Heavy Chain Variable Region QVQLVQSGAEVKKPGSSVKVSCKASGGTFNTYAINWVRQAPGQGLEWMGGIIPIFDTANYAQRFQGRVTITADKSTSTAYMELSSLRSEDTAVYFCAKDQTGYSSTLFDYWGQGTLVTVSS Sequence ID 101 HCDR2 by Kabat GIIPIFDTANYAQRFQG Sequence ID 102 HCDR3 by Kabat DQTGYSSTLFDY Sequence ID 103 Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTSHAMNWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAIDRGYMSNWVFAEYFPHWGQGTLVTVSS Sequence ID 104 Heavy Chain Variable Region EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAISWVRQAPGQGLEWMGWIIPIFDTGNYAQKIQGRVTITADKSTSTAYMELTSLRSEDTAVYYCARHDYTNTVDAFDIWGQGTMVTVSS Sequence ID 105 HCDR2 by Kabat WIIPIFDTGNYAQKIQG Sequence ID 106 HCDR3 by Kabat HDYTNTVDAFDI Sequence ID 107 Heavy Chain Variable Region QVQLVQSGAEVKKPGSSVKVSCKASGDTFRSYGITWVRQAPGQGLEWMGGIIPVFGTTNYAQKFQGRVTITADKSTSTVFMELNSLRSEDTAVYYCARRRGYSNPHWLDPWGQGTLVTVSS Sequence ID 108 HCDR2 by Kabat GIIPVFGTTNYAQKFQG Amino acid sequence of the human common light chain IGKV1-39 / jk1 (SEQ ID NO: 109) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Amino acid sequence of the common light chain variable domain of SEQ ID NO: 110 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK Amino acid sequence of the common light chain constant domain of SEQ ID NO: 111 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Amino acid sequence of SEQ ID NO: 112 CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV Amino acid sequence of Sequence ID No. 113 Hinge EPKSCDKTHTCPPCP Amino acid sequence of CH2 (SEQ ID NO: 114) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK Amino acid sequence of CH3 with the KK mutation (SEQ ID NO: 115) GQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of CH3 with the DE mutation (SEQ ID NO: 116) GQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of CD137 according to SEQ ID NO: 117 HGNC:11924 MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFS SAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCK QGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG PSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL Amino acid sequence of pembrolizumab heavy chain (SEQ ID NO: 118) QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Amino acid sequence of pembrolizumab light chain (SEQ ID NO: 119) EIVLTQSPATLLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A pharmaceutical composition for treating cancer that has relapsed to PD-1 / PD-L1 therapy and / or is positive for PD-L1 expression, wherein the pharmaceutical composition comprises an antigen-binding site that binds to the extracellular portion of CD137, comprising a heavy chain variable domain comprising CDR1, 2, and 3 of SEQ ID NO: 49 and a light chain variable domain comprising LCDR1 comprising the amino acid sequence QSISSY, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence QQSYSTPPT; and an antigen-binding site that binds to the extracellular portion of PD-L1, comprising a heavy chain variable domain comprising CDR1, 2, and 3 of SEQ ID NO: 67 and a light chain variable domain comprising LCDR1 comprising the amino acid sequence QSISSY, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence QQSYSTPPT. A pharmaceutical composition which is administered in combination with pembrolizumab.

2. A pharmaceutical composition comprising pembrolizumab for treating cancer that has relapsed to PD-1 / PD-L1 therapy and / or is positive for PD-L1 expression, The pharmaceutical composition is administered in combination with a multispecific antibody, the pharmaceutical composition comprising: an antigen-binding site that binds to the extracellular portion of CD137, comprising a heavy chain variable domain comprising CDR1, 2, and 3 of SEQ ID NO: 49, and a light chain variable domain comprising LCDR1 comprising the amino acid sequence QSISSY, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence QQSYSTPPT; and an antigen-binding site that binds to the extracellular portion of PD-L1, comprising a heavy chain variable domain comprising CDR1, 2, and 3 of SEQ ID NO: 67, and a light chain variable domain comprising LCDR1 comprising the amino acid sequence QSISSY, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence QQSYSTPPT.

3. The pharmaceutical composition according to claim 1 or 2, wherein the multispecific antibody is administered simultaneously with pembrolizumab, sequentially, or separately.

4. The pharmaceutical composition according to claim 1 or 2, wherein the cancer is a progressive or metastatic solid tumor.

5. The pharmaceutical composition according to claim 1 or 2, wherein the cancer is selected from locally advanced or metastatic lung cancer and locally advanced or metastatic melanoma.

6. The pharmaceutical composition according to claim 5, wherein the cancer is NSCLC.

7. The pharmaceutical composition according to claim 5, wherein the melanoma is selected from cutaneous melanoma, acral melanoma, or mucosal melanoma.

8. The pharmaceutical composition according to claim 5, wherein the cancer is Merkel cell carcinoma (MCC).

9. The pharmaceutical composition according to claim 1 or 2, wherein the cancer is present in humans.

10. The pharmaceutical composition according to claim 1 or 2, wherein the multispecific antibody is administered in a dose of 10 mg to 1200 mg.

11. The pharmaceutical composition according to claim 1 or 2, wherein the multispecific antibody is administered in doses of 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, or 75 mg.

12. The pharmaceutical composition according to claim 1 or 2, wherein pembrolizumab is administered in a dose of 300 to 500 mg.

13. The pharmaceutical composition according to claim 1 or 2, wherein pembrolizumab is administered in a dose of 100 to 300 mg.

14. The pharmaceutical composition according to claim 1 or 2, wherein pembrolizumab is administered every 2, 3, 4, 5, 6, 7, or 8 weeks.

15. The pharmaceutical composition according to claim 1 or 2, wherein the multispecific antibody is administered once every two weeks.

16. The pharmaceutical composition according to claim 1 or 2, wherein the multispecific antibody is administered once every three weeks.

17. The multispecific antibody is The heavy chain variable domain includes CDR1 having the amino acid sequence shown in SEQ ID NO: 50, CDR2 having the amino acid sequence shown in SEQ ID NO: 51, and CDR3 having the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable domain includes LCDR1 having the amino acid sequence QSISSY, LCDR2 having the amino acid sequence AAS, and LCDR3 having the amino acid sequence QQSYSTPPT, and comprises a binding domain that binds to CD137. The aforementioned antibody A heavy chain variable domain comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 68, CDR2 having the amino acid sequence shown in SEQ ID NO: 55, and CDR3 having the amino acid sequence shown in SEQ ID NO: 56, and a light chain variable domain comprising LCDR1 having the amino acid sequence QSISSY, LCDR2 having the amino acid sequence AAS, and LCDR3 having the amino acid sequence QQSYSTPPT, comprising a binding domain that binds to PD-L1, The pharmaceutical composition according to claim 1 or 2.

18. A pharmaceutical composition for treating cancer that has relapsed to PD-1 / PD-L1 therapy and / or is positive for PD-L1 expression, comprising a multispecific antibody, The heavy chain variable domain includes CDR1 having the amino acid sequence shown in SEQ ID NO: 50, CDR2 having the amino acid sequence shown in SEQ ID NO: 51, and CDR3 having the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable domain includes LCDR1 having the amino acid sequence QSISSY, LCDR2 having the amino acid sequence AAS, and LCDR3 having the amino acid sequence QQSYSTPPT, and comprises a binding domain that binds to CD137. The aforementioned antibody The heavy chain variable domain includes CDR1 having the amino acid sequence shown in SEQ ID NO: 68, CDR2 having the amino acid sequence shown in SEQ ID NO: 55, and CDR3 having the amino acid sequence shown in SEQ ID NO: 56, and the light chain variable domain includes LCDR1 having the amino acid sequence QSISSY, LCDR2 having the amino acid sequence AAS, and LCDR3 having the amino acid sequence QQSYSTPPT, and comprises a binding domain that binds to PD-L1. The pharmaceutical composition is administered simultaneously with, sequentially with, or separately from, pembrolizumab. A pharmaceutical composition wherein the cancer is selected from progressive or metastatic solid tumors.

19. The pharmaceutical composition according to claim 1, 2, or 18, wherein the multispecific antibody comprises the amino acid sequence of SEQ ID NO: 49 having 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, and the amino acid sequence of SEQ ID NO: 67 having 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, and the amino acid insertions, deletions, substitutions, or combinations thereof are not present in the amino acid sequence of the CDR region.

20. The pharmaceutical composition according to claim 1, 2, or 18, wherein the multispecific antibody comprises SEQ ID NO: 49 and SEQ ID NO:

67.

21. The pharmaceutical composition according to claim 1, 2, or 18, wherein the antibody comprises a common light chain variable domain having the amino acid sequence shown in SEQ ID NO: 110, or a common light chain having the amino acid sequence shown in SEQ ID NO:

109.

22. The pharmaceutical composition according to claim 1, 2, or 18, wherein the antibody comprises a heavy chain constant domain 1 (CH1) having the amino acid sequence shown in SEQ ID NO: 112 and having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or combinations thereof; a heavy chain constant domain 2 (CH2) having the amino acid sequence shown in SEQ ID NO: 114 and having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or combinations thereof; a heavy chain constant domain 3 (CH3) having the amino acid sequence shown in SEQ ID NO: 115 and having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or combinations thereof; and a heavy chain constant domain 3 (CH3) having the amino acid sequence shown in SEQ ID NO: 116 and having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or combinations thereof.

23. The pharmaceutical composition according to claim 1, 2, or 18, wherein the antibody comprises a heavy chain constant domain 1 (CH1) having the amino acid sequence shown in SEQ ID NO: 112, a heavy chain constant domain 2 (CH2) having the amino acid sequence shown in SEQ ID NO: 114, a heavy chain constant domain 3 (CH3) having the amino acid sequence shown in SEQ ID NO: 115, and a heavy chain constant domain 3 (CH3) having the amino acid sequence shown in SEQ ID NO:

116.

24. A drug combination comprising: an antigen-binding site that binds to the extracellular portion of CD137, comprising a heavy chain variable domain comprising CDR1, 2, and 3 of SEQ ID NO: 49, and a light chain variable domain comprising LCDR1 comprising the amino acid sequence QSISSY, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence QQSYSTPPT; and a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of PD-L1, comprising a heavy chain variable domain comprising CDR1, 2, and 3 of SEQ ID NO: 67, and a light chain variable domain comprising LCDR1 comprising the amino acid sequence QSISSY, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence QQSYSTPPT; and instructions for the use of the multispecific antibody in combination with pembrolizumab.

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